Peptide Dicer Substrate Agents And Methods For Their Specific Inhibition Of Gene Expression

Abstract

This invention relates to compounds, compositions, and methods useful for reducing a target RNA and protein levels via use of Dicer substrate siRNA (DsiRNA)-peptide conjugates.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is related to and claims priority under 35 U.S.C. .sctn.119(e) to U.S. provisional patent application No. 61/183,815, filed Jun. 3, 2009, and to U.S. provisional patent application No. 61/183,818, filed Jun. 3, 2009. The entire teachings of these applications are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates to peptide-dicer substrate conjugates and their method of use.

BACKGROUND OF THE INVENTION

[0003] Identification of peptide aptamers is important in view of a need for safe, efficient delivery of therapeutic molecules. Peptide aptamers have been described (reviewed in Beldhoen 2008 Int. J. Mol. Sci. 9:1276-1320; Moschos et al. 2007 Biochemical Society Transaction Vol. 35, pt 4: 807-810).

BRIEF SUMMARY OF THE INVENTION

[0004] The present invention is directed to compositions that contain double stranded RNA ("dsRNA") conjugated to peptides and methods for preparing them. The dsRNAs of the invention are double stranded RNA, small interfering RNA (siRNA) and Dicer substrate siRNAs ("DsiRNAs") with structures that are optimized, by conjugation to a peptide, for efficient delivery and/targeting and to act as effective and highly potent inhibitory agents, optionally possessing extended duration of inhibitory effect.

[0005] In one embodiment, the invention provides for an isolated double stranded ribonucleic acid (dsRNA) composition comprising a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus wherein said first strand and said second strand have a length that is at least 16 and at most 50 nucleotides in length, and a peptide, wherein said peptide has a net charge of about +5 or less and wherein the peptide is conjugated to said dsRNA.

[0006] In another embodiment, the invention provides for an isolated double stranded ribonucleic acid (dsRNA) composition comprising a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus wherein said first strand and said second strand have a length that is at least 16 and at most 50 nucleotides in length, and a peptide, wherein the peptide has no net charge and wherein said peptide is conjugated to said dsRNA.

[0007] In another embodiment, the invention provides for an isolated double stranded ribonucleic acid (dsRNA) composition comprising a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus wherein said first strand and said second strand have a length that is at least 16 and at most 50 nucleotides in length, and a peptide, wherein said peptide has a net charge of about +4 or less and wherein said peptide has at least one anionic amino acid residue. and wherein said peptide is conjugated to said dsRNA.

[0008] In one aspect the anionic amino acid is glutamic acid or aspartic acid.

[0009] In another embodiment, the invention provides for an isolated double stranded ribonucleic acid (dsRNA) composition comprising a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus wherein said first strand and said second strand have a length that is at least 6 and at most 19 nucleotides in length, and a peptide, wherein the peptide has a net charge of about +4 or less and wherein said peptide is conjugated to said dsRNA.

[0010] In another aspect, the first strand and said second strand have a length that is at least 25 and at most 35 nucleotides, at least 19 and at most 35 nucleotides, at least 19 and at most 24 nucleotides, at least 25 and at most 30 nucleotides, at least 26 and at most 30 nucleotides or at least 21 and a most 23 nucleotides.

[0011] In another aspect, the second strand comprises an overhang at the 3' terminus.

[0012] In another aspect, the first strand comprises an overhang at the 3' terminus.

[0013] In another aspect, at least one of said second strand and said first strand comprises an overhang at the 3' terminus.

[0014] In another aspect, the nucleotides of said 3' overhang of said first and/or second strand comprise a modified nucleotide.

[0015] In another aspect, the 3' overhang(s) is/are 1-5 nucleotides in length.

[0016] In another aspect, each of said first and second strands consists of the same number of nucleotide residues.

[0017] In another aspect the ultimate residue of said 5' terminus of said first strand and the ultimate residue of said 3' terminus of said second strand form a mismatched base pair.

[0018] In another aspect, the ultimate residue of said 3' terminus of said first strand and the ultimate residue of said 5' terminus of said second strand form a mismatched base pair.

[0019] In another aspect, the ultimate and penultimate residues of said 5' terminus of said first strand and the ultimate and penultimate residues of said 3' terminus of said second strand form two mismatched base pairs.

[0020] In another aspect, the ultimate and penultimate residues of said 3' terminus of said first strand and the ultimate and penultimate residues of said 5' terminus of said second strand form two mismatched base pairs.

[0021] In another aspect the peptide comprises 6-50 amino acids.

[0022] In another aspect, the peptide comprises 10-50 amino acids.

[0023] In another aspect, the peptide comprises 15-30 amino acids.

[0024] In another aspect, the peptide comprises up to 10 amino acids.

[0025] In another aspect, the peptide has a net charge of about +2 or less.

[0026] In another aspect, the peptide has a net charge of about +1 or less.

[0027] In another aspect, the peptide comprises one or more proline residues.

[0028] In another aspect, the peptide comprises one or more hydrophobic amino acid residues.

[0029] In another aspect the peptide comprises five or more cationic amino acid residues.

[0030] In another aspect the peptide comprises four cationic amino acid residues.

[0031] In another aspect the peptide comprises three cationic amino acid residues.

[0032] In another aspect the peptide comprises two cationic amino acid residues.

[0033] In another aspect the peptide comprises one cationic amino acid residue.

[0034] In another aspect the peptide has no cationic amino acid residues.

[0035] In another aspect the peptide is conjugated to said dsRNA with a stable linker.

[0036] In another aspect the stable linker comprises a homobifunctional crosslinker.

[0037] In another aspect the stable linker comprises a hetero-bifunctional crosslinker In another aspect the peptide is conjugated to said dsRNA with a cleavable linker.

[0038] In another aspect the cleavable linker comprises a disulfide linker.

[0039] In another aspect the peptide is conjugated to said dsRNA with a carbon linker.

[0040] In another aspect the carbon linker comprises no more than eighteen carbons

[0041] In another aspect the carbon linker comprises 6 carbons.

[0042] In another aspect the peptide and said dsRNA are conjugated without a linker.

[0043] In another aspect the peptide is conjugated to the 3' end of the first strand of said dsRNA.

[0044] In another aspect the peptide is conjugated to the 3' end of said second strand of said dsRNA.

[0045] In another aspect the peptide is conjugated to the 5' end of the first strand of said dsRNA.

[0046] In another aspect the peptide is conjugated to the 5' end of said second strand of said dsRNA.

[0047] In another aspect the peptide is conjugated to the 5' end of the first strand and the 5' end of said second strand of said dsRNA.

[0048] In another aspect the peptide is conjugated to the 5' end of said first strand and said 3' end of said second strand of said dsRNA.

[0049] In another aspect the peptide is conjugated to the 3' end of the first strand and the 3' end of said second strand of said dsRNA.

[0050] In another aspect the peptide is conjugated to the 3' end of said first strand and said 5' end of said second strand of said dsRNA.

[0051] In another aspect the at least one peptide is conjugated internally to said first strand of said dsRNA.

[0052] In another aspect the at least one peptide is conjugated internally to said second strand of said dsRNA.

[0053] In another aspect at least one peptide is conjugated internally to said first strand and at least one peptide is conjugated internally to said second strand of said dsRNA.

[0054] In another aspect the at least two peptides are conjugated to said dsRNA.

[0055] In another aspect the at least two peptides are identical.

[0056] In another aspect the at least two peptides are not identical.

[0057] In another aspect the isolated composition further comprises at least one dye molecule, and wherein said dye molecule is conjugated to at least one of said dsRNA and said peptide.

[0058] In another aspect the dye molecule is polyaromatic.

[0059] In another aspect the dye is a fluorescent dye.

[0060] In another aspect the isolated composition further comprises a therapeutic agent.

[0061] In another aspect the therapeutic agent is an anticancer drug.

[0062] In another aspect the anticancer drug is selected from the group consisting of paclitaxel, tamoxifen, cisplatin, doxorubicin and vinblastine.

[0063] In another aspect the therapeutic agent is a drug to treat a metabolic disease or disorder.

[0064] In another aspect the isolated composition further comprises at least one targeting peptide.

[0065] In another aspect the peptide comprises a portion of a translocation domain of a toxin.

[0066] In another aspect the neurotoxin is a clostridial neurotoxin.

[0067] In another aspect wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand of said dsRNA, position 1, 2 and/or 3 is/are substituted with a modified nucleotide.

[0068] In another aspect the modified nucleotide is a deoxyribonucleotide.

[0069] In another aspect one or both of the first and second oligonucleotide strands comprises a 5' phosphate.

[0070] In another aspect the at least one nucleotide of said first or second strand is modified.

[0071] In another aspect the modified nucleotide residues are selected from the group consisting of 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, 2'-amino and 2'-O--(N-methylcarbamate).

[0072] In another aspect the dsRNA is cleaved endogenously in said cell by Dicer.

[0073] In another aspect the amount of said isolated double stranded nucleic acid sufficient to reduce expression of the target gene is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less and 10 picomolar or less in the environment of said cell.

[0074] In another aspect the first and second strands are joined by a chemical linker.

[0075] In another aspect the 3' terminus of said first strand and said 5' terminus of said second strand are joined by a chemical linker.

[0076] In another aspect a nucleotide of said second or first strand is substituted with a modified nucleotide that directs the orientation of Dicer cleavage.

[0077] In another aspect the isolated composition comprises a modified nucleotide selected from the group consisting of a deoxyribonucleotide, a dideoxyribonucleotide, an acyclonucleotide, a 3'-deoxyadenosine (cordycepin), a 3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxyinosine (ddI), a 2',3'-dideoxy-3'-thiacytidine (3TC), a 2',3'-didehydro-2',3'-dideoxythymidine (d4T), a monophosphate nucleotide of 3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxy-3'-thiacytidine (3TC) and a monophosphate nucleotide of 2',3'-didehydro-2',3'-dideoxythymidine (d4T), a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2'-O-alkyl ribonucleotide, a 2'-O-methyl ribonucleotide, a 2'-amino ribonucleotide, a 2'-fluoro ribonucleotide, and a locked nucleic acid.

[0078] In another aspect the isolated composition comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.

[0079] In another aspect the modified nucleotide residue of said 3' terminus of said first strand is selected from the group consisting of a deoxyribonucleotide, an acyclonucleotide and a fluorescent molecule.

[0080] In another aspect the at least one of said nucleotides of said first strand and at least one of said nucleotides of said second strand form a mismatched base pair.

[0081] In another aspect the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-89.

[0082] In another embodiment the invention provides for a method for reducing expression of a target gene in a cell, comprising: contacting a cell with an isolated composition of the invention, in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA.

[0083] In another embodiment, the invention provides for a method for selectively inhibiting the growth of a cell comprising contacting a cell with an amount of said an isolated composition of the invention sufficient to inhibit the growth of the cell.

[0084] In another embodiment, the invention provides for a method for reducing expression of a target gene in an animal, comprising: treating an animal with an isolated composition of the invention, in an amount effective to reduce expression of a target gene in a cell of the animal in comparison to a reference dsRNA.

[0085] In one aspect the isolated composition possesses enhanced pharmacokinetics when compared to an appropriate control dsRNA.

[0086] In another aspect the dsRNA possesses enhanced pharmacodynamics when compared to an appropriate control dsRNA.

[0087] In another aspect the dsRNA possesses reduced toxicity when compared to an appropriate control dsRNA.

[0088] In another aspect the dsRNA possesses enhanced intracellular uptake when compared to an appropriate control dsRNA.

[0089] In another embodiment, the invention provides for a pharmaceutical composition for reducing expression of a target gene in a cell of a subject comprising an isolated composition of the invention in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA and a pharmaceutically acceptable carrier.

[0090] In another embodiment, the invention provides for a method of synthesizing a dsRNA-peptide conjugate of the invention, comprising chemically or enzymatically synthesizing said dsRNA.

[0091] In another embodiment, the invention provides for a kit comprising a dsRNA-peptide conjugate of the invention and instructions for its use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0092] FIG. 1 (A-H) presents exemplary structures of dsRNA-peptide conjugates useful according to the invention. "P"=a peptide according to the invention (A-blunt-blunt), (B and C-blunt-overhang), (D and E-asymmetric) and (F and G-mismatched ends).

[0093] FIG. 2 shows exemplary sequences of HPRT1- and KRAS-targeting dsRNAs of the invention. Underlined residues indicate positions of 2'-O-methyl modifications. Arrows indicate projected sites of dicer enzyme cleavage within the dsRNAs, while dashed lines indicate the projected position of Argonaute2-mediated cleavage within a corresponding target RNA sequence.

[0094] FIG. 3 shows exemplary peptide sequences of the peptide-conjugated dsRNAs of the invention. Net charge of each peptide is also listed.

[0095] FIG. 4 schematically depicts exemplary DsiRNA-peptide conjugates of the invention, with size shifts of properly conjugated molecules shown in lanes 2, 3 and 5 for respective DsiRNA-peptide conjugates numbered 2, 3 and 5. Arrowheads in schematics indicate projected dicer enzyme cleavage sites within the DsiRNA and DsiRNA-peptide conjugates.

[0096] FIG. 5 schematically depicts additional exemplary DsiRNA-peptide conjugates of the invention, with size shifts of properly conjugated molecules shown in lanes 2, 3, 5 and 6 for respective DsiRNA-peptide conjugates numbered 2, 3, 5 and 6. Arrowheads in schematics indicate projected dicer enzyme cleavage sites within the DsiRNA and DsiRNA-peptide conjugates.

[0097] FIG. 6 schematically depicts further exemplary DsiRNA-peptide conjugates of the invention, with size shifts indicating properly conjugated molecules shown in lanes 2 and 3 for the DsiRNA-peptide conjugates numbered 2 and 3. Arrowheads in schematics indicate projected dicer enzyme cleavage sites within the DsiRNA and DsiRNA-peptide conjugates.

[0098] FIG. 7 schematically depicts exemplary DsiRNA-peptide conjugates, including cleavable peptide conjugates, of the invention, with size shifts of properly conjugated molecules shown in lanes 2, 3, 4 and 5 for respective DsiRNA-peptide conjugates numbered 2, 3, 4 and 5. Arrowheads in schematics indicate projected dicer enzyme cleavage sites within the DsiRNA and DsiRNA-peptide conjugates.

[0099] FIG. 8 schematically depicts an exemplary DsiRNA-cyclic peptide conjugate of the invention, with a size shift indicating a properly conjugated molecule shown in lane 2 for the DsiRNA-peptide conjugate numbered 2. Arrowheads in schematics indicate projected dicer enzyme cleavage sites within the DsiRNA and DsiRNA-peptide conjugates.

[0100] FIGS. 9 and 10 schematically depict exemplary DsiRNA-peptide conjugates of the invention, with each figure showing results of Dicer processing assays for DsiRNA and DsiRNA-peptide conjugates.

[0101] FIGS. 11 and 12 show histogram data demonstrating that transfected DsiRNA-peptide conjugates were effective gene silencing agents that retained potency in vitro. Transfection assays were performed in HeLa cells.

[0102] FIGS. 13 and 14 demonstrate serum stability of exemplary DsiRNA-peptide conjugates, with half-lives indicated.

[0103] FIGS. 15, 16 and 17 show histogram data demonstrating that exemplary DsiRNA-peptide conjugates showed target gene silencing efficacy in vitro in the absence of transfection vehicle, with improved delivery observed with increasing DsiRNA-peptide conjugate concentration. Assays were performed in HeLa cells.

[0104] FIG. 18 shows histogram data demonstrating that exemplary DsiRNAs and DsiRNA-peptide conjugates knocked down target gene in HepG2 cells in vitro, in the absence of transfection vehicle. DsiRNA, DsiRNA-peptides and peptides were administered at 5 .mu.M concentrations.

[0105] FIG. 19 shows IC.sub.50 curve data demonstrating that exemplary DsiRNAs and DsiRNA-peptide conjugates knocked down target gene in HepG2 cells in vitro, in the absence of transfection vehicle. Schematics of tested agents are also shown.

DETAILED DESCRIPTION OF THE INVENTION

[0106] The present invention is directed to compositions that contain double stranded RNA ("dsRNA") comprising a peptide capable of enhancing the delivery and/or biodistribution or targeting of a dsRNA to a target and adding further functionality and/or enhancing, e.g. pharmacokinetics or pharmacodynamics of such agents as compared to dsRNA molecules that do not comprise a peptide as described herein. The present invention is also directed to methods of preparing dsRNAs comprising a peptide that are capable of reducing the level and/or expression of genes in vivo or in vitro.

[0107] The invention provides for novel dsRNA peptide conjugates.

[0108] The invention also provides for novel dsRNA-peptide conjugates for targeting dsRNA to a specific tissue. The peptide based targeting described herein occurs via highly specific binding of the targeting peptide to a surface marker on a tissue or tumor of interest. This specificity of peptide binding provides the dsRNA-peptide conjugates of the invention with an increased ability to target the dsRNA to a target in a highly specific, selective and efficient manner that is advantageous to dsRNA targeting methods or agents known in the art.

[0109] The invention provides the following advantages. The invention provides for delivery peptides that enhance delivery of a dsRNA of the invention. The invention provides for delivery peptides that are close to neutral or are neutral. Nucleic acids conjugated to cationic peptides, for example. TAT) (Tat.sup.48-60), penetratin (Antp.sup.43-58, oligoarginine (R8, R9), etc.) are known in the art. Unlike the peptides of the invention which are neutral or close to neutral, cationic peptide conjugation is especially disadvantageous for dsRNA conjugation due to the polyanionic nature of nucleic acids.

[0110] The peptides of the invention are also advantageous over the peptides known in the art because the peptides described herein, do not need to be linked to the dsRNA via a cleavable linker but can be conjugated to a dsRNA via a stable linker, since dicer enzyme will process the dsRNA-peptides of the invention to produce the siRNA molecule suitable for processing in the RISC pathway. This is especially advantageous for pharmaceutical compositions due to improved stability of stable linkers (cleavable linkers may cleave during manufacturing and/or shelf storage thereby losing their functionality).

DEFINITIONS

[0111] The invention provides improved compositions and methods for reducing expression of a target gene in a cell, involving contacting a target, with an isolated dsRNA in an amount effective to reduce expression of a target gene in a cell. The dsRNA molecules of the invention comprise a peptide, as defined herein to provide a dsRNA-peptide conjugate. The peptide enhances the delivery and/or biodistribution or targeting of a dsRNA to a target RNA and add further functionality, e.g. pharmacokinetics or pharmacodynamics as compared to dsRNA agents of corresponding length that do not contain a pattern of modified nucleotides.

[0112] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0113] The present invention features one or more dsRNA molecules conjugated to one or more peptides according to the invention and methods of using these dsRNA molecules to modulate the levels of an RNA or encoded protein of interest.

[0114] A dsRNA-peptide of the invention can be cleaved by dicer and can inhibit expression of a target RNA.

[0115] A "peptide" as used herein includes a "delivery peptide" and a "targeting peptide."

[0116] A "peptide" as used herein means a linear peptide, a branched peptide or a cyclic peptide.

[0117] The present invention further relates to the use of a peptide for transporting a dsRNA to a desired target, for example a cell or a receptor on or internal to a cell, a desired target tissue or a desired target cell.

[0118] In accordance with the present invention, the desired site may be, for example and without limitation, the brain, the adrenal or other sites outside the brain (e.g., an extracranial site) such as for example, the kidney, the liver, the pancreas, the heart, the spleen, the gastrointestinal (GI) tract (e.g., stomach, intestine, colon), the eyes, the lungs, skin, adipose, muscle, lymph nodes, bone marrow, the urinary and reproductive systems (ovary, breasts, testis, prostrate), placenta, blood cells and combination thereof. Therefore, the desired target site may be one or more site selected from the group consisting of the brain, the adrenal or other sites outside the brain (e.g., an extracranial site) such as for example, the kidney, the liver, the pancreas, the heart, the spleen, the gastrointestinal (GI) tract (e.g., stomach, intestine, colon), the eyes, the lungs, skin, adipose, muscle, lymph nodes, bone marrow, the urinary and reproductive systems (ovary, breasts, testis, prostrate), placenta, blood cells and combination thereof.

[0119] A "target cell" means any cell as defined herein, for example a cell derived from or present in any organ including but not limited to the brain, the adrenal or other sites outside the brain (e.g., an extracranial site) such as for example, the kidney, the liver, the pancreas, the heart, the spleen, the gastrointestinal (GI) tract (e.g., stomach, intestine, colon), the eyes, the lungs, skin, adipose, muscle, lymph nodes, bone marrow, the urinary and reproductive systems (ovary, breasts, testis, prostrate), placenta, blood cells and a combination thereof.

[0120] As used herein, a "delivery peptide" means a peptide that is neutral or essentially neutral. "Essentially neutral" means having a net charge of +5 or less, for example, +5, +4, +3, +2, +1 or zero.

[0121] A "net charge" according to the invention is determined according to methods known in the art. For example, the net charge as defined herein is determined by obtaining the net charge of the total number of cationic amino acids (lysine, arginine, histidine) and the total number of anionic amino acids (aspartic acid and glutamic acid.)

[0122] As used herein, "delivery peptide" means at least 6 amino acids wherein the peptide has a net charge of about +5 or less (for example, +5, +4, +3, +2, +1 or zero). In one aspect, a peptide is 6-100 amino acids, for example, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100, and has a net charge of about +5 or less. In another embodiment, a peptide is 10-50 amino acids (for example, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids) and has a net charge of about +5 or less. In another embodiment, a peptide is 15-30 amino acids (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids) and has a net charge of about +5 or less.

[0123] A "delivery peptide" according to the invention includes a peptide that is at least 6 amino acids and is a neutral peptide. In one aspect, a peptide is 6-100 amino acids, for example, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100, and has no net charge. In another embodiment, a peptide is 10-50 amino acids (for example, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids) and has no net charge. In another embodiment, a peptide is 15-30 amino acids (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids) and has no net charge.

[0124] A "delivery peptide" according to the invention also means a peptide that is at least 6 and no more than 19 amino acids, wherein the peptide has a net charge of about +5 or less (for example, +5, +4, +3, +2, +1, or zero).

[0125] As used herein, "delivery peptide" means at least 6 amino acids wherein the peptide has a net charge of about +5 or less (for example, +5, +4, +3, +2, +1 or zero) and wherein the peptide has at least one anionic amino acid. In one aspect, a peptide is 6-100 amino acids, for example, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100, and has a net charge of about +4 or less. In another embodiment, a peptide is 10-50 amino acids (for example, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids) and has a net charge of about +5 or less. In another embodiment, a peptide is 15-30 amino acids (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids) and has a net charge of about +5 or less.

[0126] As used herein, "at least one anionic amino acid" means at least one of glutamic acid (E) or aspartic acid (D). For example, XXXEXX or XXXDXX or XXDXEXX or XXXEDXX wherein X is any amino acid, wherein the peptide has a net charge of +5 or less.

[0127] A peptide that has no net charge means a "neutral peptide."

[0128] As used herein, a "neutral peptide" has a net charge that is approximately zero at neutral pH (for example pH 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4 or 8.5).

[0129] A "neutral peptide" also includes a peptide that has a net charge that is approximately zero at neutral pH and/or has an isoelectric point (pI) of about pH 7 (for example pH 6. 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4 or 8.5).

[0130] Positively charged amino acids are Lysine (Lys, K), Arginine (Arg, R) and Histidine (His, H). Negatively charged amino acids are Aspartic acid or aspartate (Asp, D), Glutamic acid or glutamate (Glu, E). (Reference: Lehninger Principles of Biochemistry, 3.sup.rd Ed., 2000. Edited by David L. Nelson and Michael M. Cox, Worth Publishers, New York, N.Y.)

[0131] A "delivery peptide" according to the invention is an amino acid sequence that can deliver a dsRNA to the appropriate target RNA when conjugated to a dsRNA of the invention.

[0132] A "delivery peptide" also means an amino acid sequence that can transport a dsRNA across a cell membrane when the dsRNA is conjugated to the peptide.

[0133] A "delivery peptide" that is useful according to the invention increases the internalization of a dsRNA to a target cell when the peptide is conjugated to the dsRNA, as compared to a dsRNA that is not conjugated to a peptide.

[0134] A "delivery peptide" that is useful according to the invention increases the delivery of a dsRNA to a target RNA when the peptide is conjugated to the dsRNA, as compared to a dsRNA that is not conjugated to a peptide.

[0135] As used herein, "increases" means delivery of a peptide-dsRNA to a target RNA is 1, 2, 3, 4, 5, 10, 15, 20, 25, 40, 35, 40, 45, 50, 100, 1000 or 10,000-fold or more greater than delivery of a dsRNA that is not conjugated to a peptide.

[0136] As used herein, "increases" means delivery of a peptide-dsRNA conjugate to a target is 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% greater than delivery of a dsRNA that is not conjugated to a peptide.

[0137] "Delivery" of a dsRNA, a peptide or a dsRNA-peptide conjugate is assessed by internalization or uptake assays described hereinbelow.

[0138] In another embodiment a "peptide" as used herein means a "targeting peptide" that is 6-100 amino acids, for example, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100, and that binds to a target cell.

[0139] A "targeting peptide" according to the invention binds specifically to a target or a binding site when conjugated to a dsRNA as defined herein.

[0140] As used herein, "specifically binds" means via hydrogen bonding or electrostatic attraction to a receptor of interest.

[0141] In one aspect, the target is a receptor or a receptor binding protein.

[0142] In one aspect, the target or binding site or receptor is on the surface of a cell.

[0143] In another aspect, the target or binding site or receptor is internal, for example, in a cell, (for example in the cytoplasm, in the nucleus or on the surface of the nucleus.)

[0144] In another aspect, the target or binding site or receptor is naked in solution.

[0145] "Specific binding" is determined by a binding assay known in the art and as defined herein (See for example US20080064092 and US2009004174). In one embodiment, specific binding is determined by comparing the binding of a dsRNA-delivery peptide to the stated, corresponding receptor to the binding of the dsRNA-peptide to other receptors, wherein all receptors are present in a mixture. An increase, as defined herein, in binding to the stated receptor, as compared to other receptors, is indicative of specific binding.

[0146] In one embodiment, specific binding is determined by comparing the binding of a dsRNA-delivery peptide to the stated cell to the binding of the dsRNA-peptide to other cells, wherein all cells are present in a mixture. An increase, as defined herein, in binding to the stated cell, as compared to other cells, is indicative of specific binding.

[0147] "Specific binding" is determined in vitro by determining the binding of a dsRNA-peptide to a naked receptor in solution or in vivo by determining the binding of a dsRNA-peptide to a cell.

[0148] As used herein, a "receptor" includes cell surface receptors, naked receptors in solution and receptors that are internal to a cell, for example in the cytoplasm, the nucleus or on the surface of the nucleus.

[0149] As used herein, a "receptor binding protein means

[0150] A "targeting peptide" as used herein, can do at least one of cross a cell membrane when conjugated to a dsRNA, transport a dsRNA across a cell membrane when conjugated to a dsRNA according to the invention and bind a receptor for the ligand, for example a cell surface receptor, when conjugated to a dsRNA.

[0151] In one aspect, a "targeting peptide" is conjugated to a translocation domain or a portion thereof, for example a translocation domain of a neurotoxin.

[0152] As used herein, a translocation domain refers to an amino acid sequence that facilitates penetration and/or internalization of a protein.

[0153] As used herein, a portion thereof means an amino acid sequence that is sufficient to maintain function, for example directing cell entry or facilitating cell surface binding, for example cell surface receptor binding, as defined herein. A "portion thereof" also means 1% or more, for example, 1, 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% of the complete amino acid sequence.

[0154] In one aspect, the targeting peptide is capable of internalization (e.g. by direct penetration or by and endocytic pathway that requires endosome formation and is also referred to as receptor-mediated endocytosis.)

[0155] "Binding" of a dsRNA, a peptide or a dsRNA-peptide conjugate is assessed by a ligand binding assay.

[0156] In one embodiment the binding affinity of the peptide or dsRNA-peptide conjugate for the corresponding receptor is about 100 nM. In another embodiment the binding affinity of the peptide or dsRNA-peptide conjugate for the corresponding receptor is about 1 nM. In another embodiment the binding affinity of the peptide or dsRNA-peptide conjugates for the corresponding receptor is about 100 nM. In another embodiment the binding affinity of the peptide or dsRNA-peptide conjugate for the corresponding receptor is about 10 nM. In another embodiment the binding affinity of the peptide or dsRNA-peptide conjugate for the corresponding receptor is about 5 nM. In another embodiment the binding affinity of the peptide or dsRNA-peptide conjugate for the corresponding receptor is about 1 nM. In another embodiment the binding affinity of the peptide or dsRNA-peptide conjugate for the corresponding receptor is about 0.1 nM or less (Gauguin et al., J Biol. Chem. 2008; 283:2604-2613; Grupping et al., Endocrinology 1997; 138(10):4064-4068; and Stone, Chervin and Kranz, Immunology. 2009; 126(2):165-76.)

[0157] In one embodiment, a "targeting peptide" means 6-100 amino acids, for example, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids, that bind to a target cell and that comprises at least a portion of an amino acid sequence of interest, for example, the amino acid sequence of a target peptide.

[0158] A peptide that is useful according to the invention increases the targeting of a dsRNA to a cell when the peptide is conjugated to the dsRNA as compared to a dsRNA that is not conjugated to a peptide.

[0159] As used herein, "increases" means targeting of a peptide-dsRNA conjugate to a cell is 1, 2, 3, 4, 5, 10, 15, 20, 25, 40, 35, 40, 45, 50, 100, 1000 or 10,000-fold or more greater than targeting of a dsRNA that is not conjugated to a peptide.

[0160] As used herein, "increases" means targeting of a peptide-dsRNA conjugate to a cell is 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% greater than targeting of a dsRNA that is not conjugated to a peptide.`

[0161] As used herein, "increases" means targeting, as defined hereinbelow, of a peptide-dsRNA conjugate to a cell requires less dsRNA (a lower dose of dsRNA) as compared to the amount or dose of an identical dsRNA that is not conjugated to a peptide and that is required to achieve an equivalent level of binding, association or internalization, as determined by the IC.sub.50s in the assays described hereinbelow. For example, the IC.sub.50 for a dsRNA-peptide conjugate that is required to achieve a 50% reduction in RNA/gene expression is decreased as compared to the IC.sub.50 for an identical dsRNA that is not conjugated to a peptide, as measured in vivo or in vitro (see for example Hefner et al. J Biomol Tech. 2008 September: 19(4) 231-237; Zimmermann et al. Nature. 2006 May 4: 441(7089):111-114; Durcan et al. Mol. Pharm. 2008 July-August; 5(4):559-566; Heidel et al. Proc Natl Acad Sci USA. 2007 Apr. 3: 104(14):5715-5721).

[0162] As used herein, "decreased" means that the IC.sub.50 for a dsRNA-peptide conjugate is 1, 2, 3, 4, 5, 10, 15, 20, 25, 40, 35, 40, 45, 50, 100, 1000 or 10,000-fold or more less than the IC.sub.50 for an identical dsRNA that is not conjugated to a peptide.

[0163] As used herein, "decreased" means that the IC.sub.50 for a dsRNA-peptide conjugate is 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% less than the IC.sub.50 for an identical dsRNA that is not conjugated to a peptide.

[0164] In one embodiment increased targeting of a dsRNA-peptide conjugate as compared to dsRNA alone as expressed by a binding coefficient, K.sub.d, is about 25%. In another embodiment the increased targeting of a dsRNA-peptide conjugate as compared to a dsRNA alone is about 100%, i.e., the dsRNA-peptide conjugate exhibits about a 2-fold increase in binding affinity (i.e., decreased K.sub.d) compared to dsRNA alone. In another embodiment the dsRNA-peptide conjugate exhibits about a 5-fold increase in binding affinity compared to dsRNA alone. In another embodiment the dsRNA-peptide conjugate exhibits about a 10-fold increase in binding affinity compared to dsiRNA alone. In another embodiment the dsRNA-peptide conjugate exhibits about a 100-fold increase in binding affinity compared to dsRNA alone. In another embodiment the dsRNA-peptide conjugate exhibits about a 1,000-fold or more increase in binding affinity compared to dsRNA alone.

[0165] "Binding" is determined by a binding assay known in the art and as defined herein. In one embodiment, binding is determined by determining the binding of a dsRNA-delivery peptide to the stated receptor.

[0166] In another embodiment, binding is determined by determining the binding of a dsRNA-delivery peptide to the stated cell wherein all cells are present in a mixture.

[0167] "Binding" is determined in vitro by determining the binding of a dsRNA-peptide to a naked receptor in solution or in vivo by determining the binding of a dsRNA-peptide to a cell.

[0168] As used herein, "targeting" means preferential or specific binding or association or internalization of a dsRNA peptide conjugate to a receptor of interest, as compared to another receptor in a mixture of receptors. As used herein "targeting" encompasses preferential or specific binding or association or internalization of a dsRNA peptide conjugate to a receptor of interest on a cell, as compared to another receptor on a cell, in a mixture of cells. As used herein "targeting" encompasses preferential or specific binding or association or internalization of a dsRNA peptide conjugate to a cell, as compared to another cell, in a mixture of cells. That is, "targeting" according to the invention, is determined or measured both in vitro and in vivo.

[0169] "Targeting" also means transport or delivery of a "peptide" of the invention to the appropriate binding site on a cell, for example, if the peptide is a ligand, targeting means delivery of the peptide to the appropriate receptor, binding or adhesion protein for the ligand.

[0170] A peptide according to the invention can be attached to the 5' or 3' end of the first strand or the 5' or 3' end of the second strand or to the 5' end of the first strand and the 5' end of the second strand, to the 5' end of the first strand and the 3' end of the second strand, to the 3' end of the first strand and the 5' end of the second strand or to the 3' end of the first strand and the 3' end of the second strand of a dsRNA of the invention.

[0171] A peptide according to the invention can also be attached internally, for example via a specific functional group on the amino acid residue (e.g., --SH group on Cys or amino group of Lys), to the first and/or second strand.

[0172] In one aspect, more than one peptide, for example a dimer, a trimer or a multitude or peptides are attached to a dsRNA.

[0173] As used herein, a "dimer" means two peptides that are conjugated to each other and wherein one of the two peptides is also conjugated to a dsRNA. A dimer also means two peptides wherein each peptide is conjugated to a unique site on a dsRNA.

[0174] As used herein, a "trimer" means three peptides that are conjugated to each other and wherein one of the three peptides is conjugated to a dsRNA. A trimer also means three peptides wherein each peptide is conjugated to a unique site on a dsRNA. A trimer also means three peptides wherein two of the three peptides are conjugated to each other and wherein one of the two peptides is also conjugated to a dsRNA and a third peptide is conjugated to a unique site on a dsRNA.

[0175] As used herein, a "multitude" means more than 1 peptide, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. The invention provides for a dsRNA that is conjugated to multiple peptides wherein the peptides are of the same or different sequences. In one embodiment, a multitude of peptides means one or more delivery peptide and one or more targeting peptide.

[0176] The term "peptide" embraces a limited number of contiguous amino acids that are peptide bonded together and comprises a targeting or delivery peptide as defined herein, whether the peptide is a naturally occurring molecule or synthetic. (i.e. a naturally occurring molecule, or a chemically/physically modified variant thereof) that is capable of delivering a dsRNA and/or binding to a peptide target, for example, a cell or a receptor on a cell.

[0177] A "peptide" as used herein can originate from a naturally occurring protein.

[0178] A "peptide" as used herein can comprise different protein domains (for example a chimeric peptide).

[0179] A "peptide" as used herein can be a synthetic peptide that is designed based on a structure-function relationship for a particular amino acid sequence and does not necessarily have homology to a natural sequence.

[0180] A peptide of the invention is conjugated to a dsRNA of the invention.

[0181] As used herein, conjugated means attached via any covalent or non-covalent association known in the art.

[0182] A peptide of the invention can be conjugated to a dsRNA of the invention via any amino acid residue in the peptide, e.g., the C-terminal amino acid of the C-terminus via the carboxyl group of the C-terminal amino acid or the N-terminal amino acid of the N-terminus via the .alpha.-amino group of the N-terminal amino acid or to a specific functional group on the amino acid residue (e.g., --SH group on Cys or amino group of Lys).

[0183] A peptide of the invention can be conjugated to a dsRNA of the invention via any amino acid residue internal in the peptide sequence, e.g., via the amino group of Lysine residues in the middle of the peptide sequence.

[0184] A peptide according to the invention can be conjugated to a dsRNA of the invention via a stable covalent linkage including but not limited to a zero-length linker, homobifunctional linker, heterobifunctional linker or a trifunctional linker (References: Bioconjugate Techniques, 1996. Greg T. Hermanson, Academic Press, San Diego, Calif.; Chemistry of Protein Conjugation and Cross-linking, 1991. Shan S. Wong, CRC Press, Boca Raton, Fla.).

[0185] As used herein, a "zero-length linker" means conjugation via a reaction where the reactants (e.g., the reactive groups on the dsRNAs and the functional groups on the peptides, such as reactive groups on the amino acid side chains, free amino and carboxyl groups of the terminal amino acid residues, etc.) are condensed to form a conjugated molecule without a linker. A "zero-length linker" is formed, for example, by reacting a terminal reactant of a peptide with the terminal reactant of a dsRNA. Examples of zero-length linking includes but are not limited to disulfides, amides, esters, thioesters, etc.

[0186] As used herein, a "homobifunctional linker" means conjugation with a linker having two similar functional groups. Examples of homobifunctional linkers include but are not limited to amino directed, carboxyl directed, sulfhydryl directed, etc.

[0187] As used herein, a "heterobifunctional linker" means conjugation with a linker having two dissimilar functional groups of different specificities. Examples of heterobifunctional linkers include but are not limited to combinations of amino and sulfhydryl directed, amino and carboxyl directed, carboxyl and sulfhydryl directed, etc.

[0188] As used herein, a "trifunctional linker" means conjugation with a linker having three reactive functional groups. Examples of trifunctional linkers include but are not limited to 4-azido-2-nitrophenylbiocytin-4-nitrophenyl ester (ABNP), sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)hexanoamido]ethy- l-1,3'-dithiopropionate (sulfo-SBED), other biocytin based molecules, etc.

[0189] A peptide according to the invention can also be conjugated to a dsRNA via a cleavable linker including but not limited to a disulfide, an ester, a glycol, a diazo, and a sulfone linker.

[0190] A peptide according to the invention can be conjugated to a dsRNA by a carbon linker, for example a carbon linker that is 1 or more carbons, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more carbons.

[0191] A peptide according to the invention can be conjugated to a dsRNA using a prosthetic group. Prosthetic groups include but are not limited to metal ions, porphyrin groups, coenzymes and other nonpeptidyl moieties, e.g., carbohydrates or oligosaccharides (Wong, S. S. (1991), Chemistry of protein conjugation and cross-linking, CRC Press).

[0192] In one embodiment, a peptide and a dsRNA are conjugated by expression as a fusion construct.

[0193] A "peptide" may be attached to a dsRNA by any conventional chemical conjugation techniques, which are well known to a skilled person. In this regard, reference is made to Hermanson, G. T. (1996), Bioconjugate techniques, Academic Press, and to Wong, S. S. (1991), Chemistry of protein conjugation and cross-linking, CRC Press.

[0194] A "peptide" may be conjugated to a dsRNA non-covalently via ionic interactions.

[0195] As used herein, a peptide-dsRNA conjugate" means a peptide that is conjugated to a dsRNA by a method including but not limited to the methods of attachment/conjugation described herein.

[0196] In one aspect a peptide-dsRNA conjugate further comprises one or more dye molecules.

[0197] As used herein, a "dye molecule" includes but is not limited to a polyaromatic dye or a fluorescent dye, for example Cy3, Cy5, Cy5.5, Alexa Fluor.RTM. (e.g, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, etc.)

[0198] In one aspect, a peptide-dsRNA conjugate further comprises a delivery peptide, as defined herein.

[0199] In one aspect, a peptide-dsRNA conjugate further comprises a therapeutic agent, for example, an anticancer agent or an agent that treats a metabolic disease or disorder. Anticancer agents include but are not limited to antiviral agents (Fiume et al. FEBS Lett. 1983; 153(1):6-10), cisplatin (Mukhopadhyay S et al., Bioconjug Chem. 2008; 19(1):39-49), doxorubicin (Guan H et al., Bioconjug Chem. 2008; 19(9):1813-21), paclitaxel (Dubikovskaya E A et al., Proc Natl Acad Sci USA. 2008; 105(34):12128-33, Regina A et al., Br J. Pharmacol. 2008; 155(2):185-97), tamoxifen (Rickert et al. Biomacromolecules. 2007; 8(11):3608-3612) and vinblastine (DeFeo-Jones D et al., Mol Cancer Ther. 2002; 1(7):451-459).

[0200] A "peptide-dsRNA conjugate" refers to a molecule wherein both of said peptide and said dsRNA retain their function.

[0201] As used herein, "decreased" for example, a decrease in the onset of action of a dsRNA-peptide conjugate, or a decrease in the speed of delivery of a dsRNA-peptide conjugate, means 1, 2, 3, 4, 5, 10, 15, 20, 25, 40, 35, 40, 45, 50, 100, 1000 or 10,000-fold or more less than the onset of action or speed of delivery of an identical dsRNA that is not conjugated to a peptide.

[0202] As used herein, "decreased" for example, a decrease in the onset of action of a dsRNA-peptide conjugate, or a decrease in the speed of delivery of a dsRNA-peptide conjugate, means 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% less than an the onset of action or speed of delivery of identical dsRNA that is not conjugated to a peptide.

[0203] As used herein, "onset of action" means the time period between the administration of a dsRNA in vitro (for example to a cell or to tissue culture medium) or in vivo (for example to a human or animal (e.g. mouse or rat) subject) and the arrival of the dsRNA at the target RNA.

[0204] As used herein, "speed of delivery" means the time required for a dsRNA to reach a target RNA following administration of a dsRNA.

[0205] As used herein, "duration of action" means the time period during which dsRNA inhibits expression of a target RNA.

[0206] As used herein, a "control" or a "reference", for example a control dsRNA, means a dsRNA that is comparable in length to the dsRNA that is specific for a particular target RNA (the test dsRNA), but that is not specific for a particular target RNA. A control RNA has a nucleotide sequence that is not identical to the dsRNA that is specific for a target of interest. A control, for example a control peptide means a peptide that is comparable in one or more of length and charge but has an amino acid sequence that is different from the amino acid sequence of the peptide that is conjugated to a dsRNA that is specific for a target RNA (the test peptide). A control, for example a control dsRNA-peptide conjugate means a dsRNA-peptide conjugate wherein the dsRNA is comparable in length to the dsRNA that is specific for a particular target RNA, but is not specific for a particular target RNA. A control dsRNA-peptide conjugate also means a dsRNA-peptide conjugate wherein the peptide is comparable in one or more of length and charge but has an amino acid sequence that is different from the amino acid sequence of the peptide that is conjugated to a dsRNA that is specific for a target RNA. A control dsRNA-peptide conjugate also means a dsRNA-peptide conjugate wherein the peptide is comparable in one or more of length and charge but has an amino acid sequence that is different from the amino acid sequence of the peptide that is conjugated to a dsRNA that is specific for a target RNA and wherein the dsRNA is comparable in length to the dsRNA that is specific for a particular target RNA, but that is not specific for a particular target RNA.

[0207] As used herein, a test peptide or a test dsRNA means a peptide or dsRNA that comprises a conjugate that decreases the expression of a target RNA according to the invention. A test dsRNA means a dsRNA that decreases the expression of a target RNA according to the invention. A "test" dsRNA-peptide conjugate comprises a test dsRNA conjugated to a test peptide.

[0208] As used herein, the term "nucleic acid" refers to deoxyribonucleotides, ribonucleotides, or modified nucleotides, and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0209] As used herein, "nucleotide" is used as recognized in the art to include those with natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, e.g., Usman and McSwiggen, supra; Eckstein, et al., International PCT Publication No. WO 92/07065; Usman et al, International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach, et al, Nucleic Acids Res. 22:2183, 1994. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, hypoxanthine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra). By "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents.

[0210] As used herein, "modified nucleotide" refers to a nucleotide that has one or more modifications to the nucleoside, the nucleobase, pentose ring, or phosphate group. For example, modified nucleotides exclude ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. Modifications include those naturally occurring that result from modification by enzymes that modify nucleotides, such as methyltransferases. Modified nucleotides also include synthetic or non-naturally occurring nucleotides. Synthetic or non-naturally occurring modifications in nucleotides include those with 2' modifications, e.g., 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH.sub.2--O-2'-bridge, 4'-(CH.sub.2).sub.2--O-2'-bridge, 2'-LNA, and 2'-O--(N-methylcarbamate) or those comprising base analogs. In connection with 2'-modified nucleotides as described for the present disclosure, by "amino" is meant 2'-NH.sub.2 or 2'-O--NH.sub.2, which can be modified or unmodified. Such modified groups are described, e.g., in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878.

[0211] In reference to the nucleic acid molecules of the present disclosure, modifications may exist upon these agents in patterns on one or both strands of the dsRNA). As used herein, "alternating positions" refers to a pattern where every other nucleotide is a modified nucleotide or there is an unmodified nucleotide (e.g., an unmodified ribonucleotide) between every modified nucleotide over a defined length of a strand of the dsRNA (e.g., 5'-MNMNMN-3'; 3'-MNMNMN-5'; where M is a modified nucleotide and N is an unmodified nucleotide). In certain embodiments, the modification pattern starts from the first nucleotide position at either the 5' or 3' terminus according to any of the position numbering conventions described herein (in certain embodiments, position 1 is designated in reference to the terminal residue of a strand following a projected Dicer cleavage event of a DsiRNA agent of the invention; thus, position 1 does not always constitute a 3' terminal or 5' terminal residue of a pre-processed agent of the invention). In other embodiments, position 1 is designated in reference to the nucleotide residue of a first or second strand that is complementary to the 5' or 3' end of the opposite strand. For example, in certain embodiments, position 1 is the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand. The invention encompasses dsRNAs wherein the modification pattern starts at any one of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 12, 18, 19, 20, 21, 22, 23 or 24 from the 5' or 3' terminus according to any of the position numbering conventions described herein. The invention also encompasses dsRNAs wherein the modification patterns starts at any position that is at least one nucleotide from the 5' or 3' terminal residue.

[0212] The pattern of modified nucleotides at alternating positions may run the full length of the strand, but in certain embodiments includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more nucleotides containing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more modified nucleotides, respectively.

[0213] As used herein, "alternating pairs of positions" refers to a pattern where two consecutive modified nucleotides are separated by two consecutive unmodified nucleotides over a defined length of a strand of the dsRNA (e.g., 5'-MMNNMMNNMMNN-3'; 3'-MMNNMMNNMMNN-5'; where M is a modified nucleotide and N is an unmodified nucleotide). In one embodiment, the modification pattern starts from the first nucleotide position at either the 5' or 3' terminus according to any of the position numbering conventions described herein. The pattern of modified nucleotides at alternating positions may run the full length of the strand, but preferably includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 nucleotides containing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 modified nucleotides, respectively. It is emphasized that the above modification patterns are exemplary and are not intended as limitations on the scope of the invention.

[0214] As used herein, "base analog" refers to a heterocyclic moiety which is located at the 1' position of a nucleotide sugar moiety in a modified nucleotide that can be incorporated into a nucleic acid duplex (or the equivalent position in a nucleotide sugar moiety substitution that can be incorporated into a nucleic acid duplex). In the dsNAs of the invention, a base analog is generally either a purine or pyrimidine base excluding the common bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U). Base analogs can duplex with other bases or base analogs in dsRNAs. Base analogs include those useful in the compounds and methods of the invention, e.g., those disclosed in U.S. Pat. Nos. 5,432,272 and 6,001,983 to Benner and US Patent Publication No. 20080213891 to Manoharan, which are herein incorporated by reference. Non-limiting examples of bases include hypoxanthine (I), xanthine (X), 313-D-ribofuranosyl-(2,6-diaminopyrimidine) (K), 3-.beta.-D-ribofuranosyl-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-d- ione) (P), iso-cytosine (iso-C), iso-guanine (iso-G), 1-.beta.-D-ribofuranosyl-(5-nitroindole), 1-.beta.-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2-aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa), 2-amino-6-(2-thienyl)purine (S), 2-oxopyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methyl isocarbostyrilyl, 5-methyl isocarbostyrilyl, and 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl, tetracenyl, pentacenyl, and structural derivates thereof (Schweitzer et al., J. Org. Chem., 59:7238-7242 (1994); Berger et al., Nucleic Acids Research, 28(15):2911-2914 (2000); Moran et al., J. Am. Chem. Soc., 119:2056-2057 (1997); Morales et al., J. Am. Chem. Soc., 121:2323-2324 (1999); Guckian et al., J. Am. Chem. Soc., 118:8182-8183 (1996); Morales et al., J. Am. Chem. Soc., 122(6):1001-1007 (2000); McMinn et al., J. Am. Chem. Soc., 121:11585-11586 (1999); Guckian et al., J. Org. Chem., 63:9652-9656 (1998); Moran et al., Proc. Natl. Acad. Sci., 94:10506-10511 (1997); Das et al., J. Chem. Soc., Perkin Trans., 1:197-206 (2002); Shibata et al., J. Chem. Soc., Perkin Trans., 1: 1605-1611 (2001); Wu et al., J. Am. Chem. Soc., 122(32):7621-7632 (2000); O'Neill et al., J. Org. Chem., 67:5869-5875 (2002); Chaudhuri et al., J. Am. Chem. Soc., 117:10434-10442 (1995); and U.S. Pat. No. 6,218,108.). Base analogs may also be a universal base.

[0215] As used herein, "universal base" refers to a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution, that, when present in a nucleic acid duplex, can be positioned opposite more than one type of base without altering the double helical structure (e.g., the structure of the phosphate backbone). Additionally, the universal base does not destroy the ability of the single stranded nucleic acid in which it resides to duplex to a target nucleic acid. The ability of a single stranded nucleic acid containing a universal base to duplex a target nucleic can be assayed by methods apparent to one in the art (e.g., UV absorbance, circular dichroism, gel shift, single stranded nuclease sensitivity, etc.). Additionally, conditions under which duplex formation is observed may be varied to determine duplex stability or formation, e.g., temperature, as melting temperature (Tm) correlates with the stability of nucleic acid duplexes. Compared to a reference single stranded nucleic acid that is exactly complementary to a target nucleic acid, the single stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower Tm than a duplex formed with the complementary nucleic acid. However, compared to a reference single stranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher Tm than a duplex formed with the nucleic acid having the mismatched base.

[0216] Some universal bases are capable of base pairing by forming hydrogen bonds between the universal base and all of the bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U) under base pair forming conditions. A universal base is not a base that forms a base pair with only one single complementary base. In a duplex, a universal base may form no hydrogen bonds, one hydrogen bond, or more than one hydrogen bond with each of G, C, A, T, and U opposite to it on the opposite strand of a duplex. Preferably, the universal bases does not interact with the base opposite to it on the opposite strand of a duplex. In a duplex, base pairing between a universal base occurs without altering the double helical structure of the phosphate backbone. A universal base may also interact with bases in adjacent nucleotides on the same nucleic acid strand by stacking interactions. Such stacking interactions stabilize the duplex, especially in situations where the universal base does not form any hydrogen bonds with the base positioned opposite to it on the opposite strand of the duplex. Non-limiting examples of universal-binding nucleotides include inosine, 1-.beta.-D-ribofuranosyl-5-nitroindole, and/or 1-.beta.-D-ribofuranosyl-3-nitropyrrole (US Pat. Appl. Publ. No. 20070254362 to Quay et al.; Van Aerschot et al., An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995 Nov. 11; 23(21):4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR. Nucleic Acids Res. 1995 Jul. 11; 23(13):2361-6; Loakes and Brown, 5-Nitroindole as an universal base analogue. Nucleic Acids Res. 1994 Oct. 11; 22(20):4039-43).

[0217] As used herein, "loop" refers to a structure formed by a single strand of a nucleic acid, in which complementary regions that flank a particular single stranded nucleotide region hybridize in a way that the single stranded nucleotide region between the complementary regions is excluded from duplex formation or Watson-Crick base pairing. A loop is a single stranded nucleotide region of any length. Examples of loops include the unpaired nucleotides present in such structures as hairpins, stem loops, or extended loops.

[0218] As used herein, "extended loop" in the context of a dsRNA refers to a single stranded loop and in addition 1, 2, 3, 4, 5, 6 or up to 20 base pairs or duplexes flanking the loop. In an extended loop, nucleotides that flank the loop on the 5' side form a duplex with nucleotides that flank the loop on the 3' side. An extended loop may form a hairpin or stem loop.

[0219] As used herein, "tetraloop" in the context of a dsRNA refers to a loop (a single stranded region) consisting of four nucleotides that forms a stable secondary structure that contributes to the stability of an adjacent Watson-Crick hybridized nucleotides. Without being limited to theory, a tetraloop may stabilize an adjacent Watson-Crick base pair by stacking interactions. In addition, interactions among the four nucleotides in a tetraloop include but are not limited to non-Watson-Crick base pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., Nature 1990 Aug. 16; 346(6285):680-2; Heus and Pardi, Science 1991 Jul. 12; 253(5016):191-4). A tetraloop confers an increase in the melting temperature (Tm) of an adjacent duplex that is higher than expected from a simple model loop sequence consisting of four random bases. For example, a tetraloop can confer a melting temperature of at least 55.degree. C. in 10 mM NaHPO.sub.4 to a hairpin comprising a duplex of at least 2 base pairs in length. A tetraloop may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Examples of RNA tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop. (Woese et al., Proc Natl Acad Sci USA. 1990 November; 87(21):8467-71; Antao et al., Nucleic Acids Res. 1991 Nov. 11; 19(21):5901-5). Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA), the d(GNRA)) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, the d(TNCG) family of tetraloops (e.g., d(TTCG)). (Nakano et al. Biochemistry, 41 (48), 14281-14292, 2002; SHINJI et al. Nippon Kagakkai Koen Yokoshu VOL. 78th; NO. 2; PAGE. 731 (2000).)

[0220] The dsRNA compositions of the invention, because they are modeled to enter the RNAi pathway as substrates of the Dicer enzyme, at least in part due the strand lengths of such compositions, are also referred to as Dicer substrate siRNA ("DsiRNA") agents herein. The "DsiRNA agent" compositions of the instant invention comprise dsRNA which is a precursor molecule for Dicer enzyme processing, i.e., the DsiRNA of the present invention is processed in vivo to produce an active siRNA. Specifically, the DsiRNA is processed by Dicer to an active siRNA which is incorporated into RISC. This precursor molecule, primarily referred to as a "DsiRNA agent" or "DsiRNA molecule" herein, can also be referred to as a precursor RNAi molecule herein. As used herein, the term "active siRNA" refers to a double stranded nucleic acid in which each strand comprises RNA, RNA analog(s) or RNA and DNA. The siRNA comprises between 19 and 23 nucleotides or comprises 21 nucleotides. The active siRNA typically has 2 bp overhangs on the 3' ends of each strand such that the duplex region in the siRNA comprises 17-21 nucleotides, or 19 nucleotides.

[0221] In certain embodiments, dsRNAs of the invention include but are not limited to dsRNAs comprising first and second strands comprising between 16 and 50, 19 and 35, 19 and 24, 25 and 30, 25 and 35, 26 and 30, 21 and 23 nucleotides in length.

[0222] A DsiRNA agent of the instant invention has a length sufficient such that it is processed by Dicer to produce an siRNA. Accordingly, a suitable DsiRNA agent contains one oligonucleotide sequence, a first sequence, that is at least 25 nucleotides in length and no longer than about 35 nucleotides. This sequence of RNA can be between about 26 and 35, 26 and 34, 26 and 33, 26 and 32, 26 and 31, 26 and 30, and 26 and 29 nucleotides in length. This sequence can be about 27 or 28 nucleotides in length or 27 nucleotides in length. The second sequence of the DsiRNA agent can be any sequence that anneals to the first sequence under biological conditions, such as within the cytoplasm of a eukaryotic cell. Generally, the second oligonucleotide sequence will have at least 19 complementary base pairs with the first oligonucleotide sequence, more typically the second oligonucleotides sequence will have about 21 or more complementary base pairs, or about 25 or more complementary base pairs with the first oligonucleotide sequence. In one embodiment, the second sequence is the same length as the first sequence, and the DsiRNA agent is blunt ended. In another embodiment, the ends of the DsiRNA agent have one or more overhangs. In certain embodiments, wherein the second sequence is the same length as the first sequence, the ultimate residue of said 3' terminus of said first strand and the ultimate residue of the said 5' terminus of the second strand form a mismatched base pair. In other embodiments, wherein the second sequence is the same length as the first sequence, the ultimate residue of the 5' terminus of said first strand and the ultimate residue of the 3' terminus of the second strand form a mismatched base pair. In other embodiments, wherein the second sequence is the same length as the first sequence, the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form two mismatched base pairs. In still other embodiments, wherein the second sequence is the same length as the first sequence, the ultimate and penultimate residues of the 5' terminus of the first strand and the ultimate and penultimate residues of the 3' terminus of the second strand form two mismatched base pairs.

[0223] In certain embodiments, the first and second oligonucleotide sequences of the DsiRNA agent exist on separate oligonucleotide strands that can be and typically are chemically synthesized. In some embodiments, both strands are between 26 and 35 nucleotides in length. In other embodiments, both strands are between 25 and 30 or 26 and 30 nucleotides in length. In one embodiment, both strands are 27 nucleotides in length, are completely complementary and have blunt ends. In one embodiment, one or both oligonucleotide strands are capable of serving as a substrate for Dicer. In other embodiments, at least one modification is present that promotes Dicer to bind to the double-stranded RNA structure in an orientation that maximizes the double-stranded RNA structure's effectiveness in inhibiting gene expression. In certain embodiments of the instant invention, the DsiRNA agent is comprised of two oligonucleotide strands of differing lengths, with the DsiRNA possessing a blunt end at the 3' terminus of a first strand (sense strand) and a 3' overhang at the 3' terminus of a second strand (antisense strand). The DsiRNA can also contain one or more deoxyribonucleic acid (DNA) base substitutions.

[0224] Suitable DsiRNA compositions that contain two separate oligonucleotides can be chemically linked outside their annealing region by chemical linking groups. Many suitable chemical linking groups are known in the art and can be used. Suitable groups will not block Dicer activity on the DsiRNA and will not interfere with the directed destruction of the RNA transcribed from the target gene. Alternatively, the two separate oligonucleotides can be linked by a third oligonucleotide such that a hairpin structure is produced upon annealing of the two oligonucleotides making up the DsiRNA composition. The hairpin structure will not block Dicer activity on the DsiRNA and will not interfere with the directed destruction of the target RNA.

[0225] As used herein, a dsRNA, e.g., DsiRNA or siRNA, having a sequence "sufficiently complementary" to a target RNA or cDNA sequence means that the dsRNA has a sequence sufficient to trigger the destruction of the target RNA (where a cDNA sequence is recited, the RNA sequence corresponding to the recited cDNA sequence) by the RNAi machinery (e.g., the RISC complex) or process. The dsRNA molecule can be designed such that every residue of the antisense strand is complementary to a residue in the target molecule. Alternatively, substitutions can be made within the molecule to increase stability and/or enhance processing activity of said molecule. Substitutions can be made within the strand or can be made to residues at the ends of the strand. In certain embodiments, substitutions and/or modifications are made at specific residues within a DsiRNA agent. Such substitutions and/or modifications can include, e.g., deoxy-modifications at one or more residues of positions 1, 2 and 3 when numbering from the 3' terminal position of the sense strand of a DsiRNA agent; deoxy-modifications at one or more residues of positions 1, 2, 3 or 4 when numbering from the 5' terminal position of the antisense strand of a DsiRNA agent and introduction of 2'-O-alkyl (e.g., 2'-O-methyl) modifications at the 3' terminal residue of the antisense strand of DsiRNA agents, with such modifications also or alternatively being present at overhang positions of the 3' portion of the antisense strand and/or throughout the DsiRNA agent, for example at alternating residues or in pairs of residues of the antisense strand of the DsiRNA that are included within the region of a DsiRNA agent that is processed to form an active siRNA agent. The preceding modifications are offered as exemplary, and are not intended to be limiting in any manner. Further consideration of the structure of preferred DsiRNA agents, including further description of the modifications and substitutions that can be performed upon the DsiRNA agents of the instant invention, can be found below.

[0226] By "complementarity" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. In one embodiment, a DsiRNA molecule of the invention comprises about 19 to about 30 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or or more) nucleotides that are complementary to one or more target nucleic acid molecules or a portion thereof.

[0227] The phrase "duplex region" refers to the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary. For example, an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 bases on each strand are complementary or substantially complementary, such that the "duplex region" consists of 19 base pairs. The remaining base pairs may, for example, exist as 5' and 3' overhangs. Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region.

[0228] Substantial complementarity refers to complementarity between the strands such that they are capable of annealing under biological conditions. Techniques to empirically determine if two strands are capable of annealing under biological conditions are well know in the art. Alternatively, two strands can be synthesized and added together under biological conditions to determine if they anneal to one another.

[0229] Single-stranded nucleic acids that base pair over a number of bases are said to "hybridize." Hybridization is typically determined under physiological or biologically relevant conditions (e.g., intracellular: pH 7.2, 140 mM potassium ion; extracellular pH 7.4, 145 mM sodium ion). Hybridization conditions generally contain a monovalent cation and biologically acceptable buffer and may or may not contain a divalent cation, complex anions, e.g. gluconate from potassium gluconate, uncharged species such as sucrose, and inert polymers to reduce the activity of water in the sample, e.g. PEG. Such conditions include conditions under which base pairs can form.

[0230] Hybridization is measured by the temperature required to dissociate single stranded nucleic acids forming a duplex, i.e., (the melting temperature; Tm). Hybridization conditions are also conditions under which base pairs can form. Various conditions of stringency can be used to determine hybridization (see, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). Stringent temperature conditions will ordinarily include temperatures of at least about 30.degree. C., more preferably of at least about 37.degree. C., and most preferably of at least about 42.degree. C. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10.degree. C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(.degree. C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(.degree. C.)=81.5+16.6(log 10[Na+])+0.41 (% G+C)-(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1.times.SSC=0.165 M). For example, a hybridization determination buffer is shown in Table 1.

TABLE-US-00001 TABLE 1 To make 50 final conc. Vender Cat# Lot# m.w./Stock mL solution NaCl 100 mM Sigma S-5150 41K8934 5M 1 mL KCl 80 mM Sigma P-9541 70K0002 74.55 0.298 g MgCl.sub.2 8 mM Sigma M-1028 120K8933 1M 0.4 mL sucrose 2% w/v Fisher BP220-212 907105 342.3 1 g Tris-HCl 16 mM Fisher BP1757-500 12419 1M 0.8 mL NaH.sub.2PO.sub.4 1 mM Sigma S-3193 52H-029515 120.0 0.006 g EDTA 0.02 mM Sigma E-7889 110K89271 0.5M 2 .mu.L H.sub.2O Sigma W-4502 51K2359 to 50 mL pH = 7.0 at 20.degree. C. adjust with HCl

[0231] Useful variations on hybridization conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Antisense to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0232] As used herein, "oligonucleotide strand" is a single stranded nucleic acid molecule. An oligonucleotide may comprise ribonucleotides, deoxyribonucleotides, modified nucleotides (e.g., nucleotides with 2' modifications, synthetic base analogs, etc.) or combinations thereof. Such modified oligonucleotides can be preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.

[0233] Certain dsRNAs of this invention can be chimeric double-stranded ribonucleic acids (dsRNAs). "Chimeric dsRNAs" or "chimeras", in the context of this invention, are dsRNAs which contain two or more chemically distinct regions, each made up of at least one nucleotide. These dsRNAs typically contain at least one region primarily comprising ribonucleotides (optionally including modified ribonucleotides) that form a Dicer substrate siRNA ("DsiRNA") molecule. This DsiRNA region can be covalently attached to a second region comprising base paired deoxyribonucleotides (a "dsDNA region") on either flank of the ribonucleotide duplex region, which can confer one or more beneficial properties (such as, for example, increased efficacy, e.g., increased potency and/or duration of DsiRNA activity, function as a recognition domain or means of targeting a chimeric dsNA to a specific location, for example, when administered to cells in culture or to a subject, functioning as an extended region for improved attachment of functional groups, payloads, detection/detectable moieties, functioning as an extended region that allows for more desirable modifications and/or improved spacing of such modifications, etc.). This second region, e.g., comprising base paired deoxyribonucleotides may also include modified or synthetic nucleotides and/or modified or synthetic deoxyribonucleotides.

[0234] As used herein, the term "ribonucleotide" encompasses natural and synthetic, unmodified and modified ribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and/or to the linkages between ribonucleotides in the oligonucleotide. As used herein, the term "ribonucleotide" specifically excludes a deoxyribonucleotide, which is a nucleotide possessing a single proton group at the 2' ribose ring position.

[0235] As used herein, the term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and/or to the linkages between deoxyribonucleotide in the oligonucleotide. As used herein, the term "deoxyribonucleotide" also includes a modified ribonucleotide that does not permit Dicer cleavage of a dsRNA agent, e.g., a 2'-O-methyl ribonucleotide, a phosphorothioate-modified ribonucleotide residue, etc., that does not permit Dicer cleavage to occur at a bond of such a residue.

[0236] As used herein, the term "PS-NA" refers to a phosphorothioate-modified nucleotide residue. The term "PS-NA" therefore encompasses both phosphorothioate-modified ribonucleotides ("PS-RNAs") and phosphorothioate-modified deoxyribonucleotides ("PS-DNAs").

[0237] As used herein, "Dicer" refers to an endoribonuclease in the RNase III family that cleaves a dsRNA or dsRNA-containing molecule, e.g., double-stranded RNA (dsRNA) or pre-microRNA (miRNA), into double-stranded nucleic acid fragments about 19-25 nucleotides long, usually with a two-base overhang on the 3' end. With respect to the dsRNAs of the invention, the duplex formed by a dsRNA region of a dsRNA of the invention is recognized by Dicer and is a Dicer substrate on at least one strand of the duplex. Dicer catalyzes the first step in the RNA interference pathway, which consequently results in the degradation of a target RNA. The protein sequence of human Dicer is provided at the NCBI database under accession number NP 085124, hereby incorporated by reference.

[0238] Dicer "cleavage" is determined as follows (e.g., see Collingwood et al., Oligonucleotides 18:187-200 (2008)). In a Dicer cleavage assay, RNA duplexes (100 pmol) are incubated in 20 .mu.L of 20 mM Tris pH 8.0, 200 mM NaCl, 2.5 mM MgCl2 with or without 1 unit of recombinant human Dicer (Stratagene, La Jolla, Calif.) at 37.degree. C. for 18-24 hours. Samples are desalted using a Performa SR 96-well plate (Edge Biosystems, Gaithersburg, Md.). Electrospray-ionization liquid chromatography mass spectroscopy (ESI-LCMS) of duplex RNAs pre- and post-treatment with Dicer is done using an Oligo HTCS system (Novatia, Princeton, N.J.; Hail et al., 2004), which consists of a ThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processing software and Paradigm MS4 HPLC (Michrom BioResources, Auburn, Calif.). In this assay, Dicer cleavage occurs where at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% of the Dicer substrate dsRNA, (i.e., 25-30 bp, dsRNA, preferably 26-30 bp dsRNA) is cleaved to a shorter dsRNA (e.g., 19-23 bp dsRNA, preferably, 21-23 bp dsRNA).

[0239] As used herein, "Dicer cleavage site" refers to the sites at which Dicer cleaves a dsRNA (e.g., the dsRNA region of a dsRNA of the invention). Dicer contains two RNase III domains which typically cleave both the sense and antisense strands of a dsRNA. The average distance between the RNase III domains and the PAZ domain determines the length of the short double-stranded nucleic acid fragments it produces and this distance can vary (Macrae I, et al. (2006). "Structural basis for double-stranded RNA processing by Dicer". Science 311 (5758): 195-8.). Dicer is projected to cleave certain double-stranded nucleic acids of the instant invention that possess an antisense strand having a 2 nucleotide 3' overhang at a site between the 21.sup.st and 22.sup.nd nucleotides removed from the 3' terminus of the antisense strand, and at a corresponding site between the 21.sup.st and 22.sup.nd nucleotides removed from the 5' terminus of the sense strand. The projected and/or prevalent Dicer cleavage site(s) for dsRNA molecules distinct from those are known in the art or may be similarly identified via art-recognized methods, including those described in Macrae et al. Dicer cleavage of a dsRNA (e.g., DsiRNA) can result in generation of Dicer-processed siRNA lengths of 19 to 23 nucleotides in length. Indeed, in one aspect of the invention that is described in greater detail below, a double stranded DNA region is included within a dsRNA for purpose of directing prevalent Dicer excision of a typically non-preferred 19mer siRNA.

[0240] As used herein, "overhang" refers to unpaired nucleotides, in the context of a duplex having one, two, three, four or five free ends at either the 5' terminus or 3' terminus of a dsRNA. In certain embodiments, the overhang is a 3' or 5' overhang on the antisense strand or sense strand.

[0241] As used herein, the term "DmiRNA" refers to a species of Dicer substrate siRNA ("DsiRNA") that possesses at least one mismatch nucleotide within the antisense (guide) strand of the DmiRNA agent, specifically within the region of the antisense strand that functions as an RNA interference agent and is believed to hybridize with the sequence of a target RNA. Such mismatch nucleotide can exist either with respect to the sense (passenger) strand, with respect to the target RNA sequence to which the antisense strand of the DmiRNA is believed to hybridize, or with respect to both.

[0242] As used herein, the term "RNA processing" refers to processing activities performed by components of the siRNA, miRNA or RNase H pathways (e.g., Drosha, Dicer, Argonaute2 or other RISC endoribonucleases, and RNaseH), which are described in greater detail below (see "RNA Processing" section below). The term is explicitly distinguished from the post-transcriptional processes of 5' capping of RNA and degradation of RNA via non-RISC- or non-RNase H-mediated processes. Such "degradation" of an RNA can take several forms, e.g. deadenylation (removal of a 3' poly(A) tail), and/or nuclease digestion of part or all of the body of the RNA by any of several endo- or exo-nucleases (e.g., RNase III, RNase P, RNase T1, RNase A (1, 2, 3, 4/5), oligonucleotidase, etc.).

[0243] By "homologous sequence" is meant, a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors. A homologous sequence can be a nucleotide sequence that is shared by two or more non-coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect homology (e.g., 100%), as partially homologous sequences are also contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.). Indeed, design and use of the DsiRNA agents of the instant invention contemplates the possibility of using such DsiRNA agents not only against target RNAs of interest possessing perfect complementarity with the presently described DsiRNA agents, but also against target RNAs of interest possessing sequences that are, e.g., only 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc. complementary to said DsiRNA agents. Similarly, it is contemplated that the presently described DsiRNA agents of the instant invention might be readily altered by the skilled artisan to enhance the extent of complementarity between said DsiRNA agents and a target RNA of interest, e.g., of a specific allelic variant (e.g., an allele of enhanced therapeutic interest). Indeed, DsiRNA agent sequences with insertions, deletions, and single point mutations relative to the target sequence of interest can also be effective for inhibition (possibly believed to act via microRNA-like translational inhibition, rather than destruction, of targeted transcripts; accordingly, such DsiRNA agents can be termed "DmiRNAs"). Alternatively, DsiRNA agent sequences with nucleotide analog substitutions or insertions can be effective for inhibition.

[0244] Sequence identity may be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions.times.100), optionally penalizing the score for the number of gaps introduced and/or length of gaps introduced.

[0245] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (i.e., a local alignment). A preferred, non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.

[0246] In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment). A preferred, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0247] Greater than 80% sequence identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity, between the DsiRNA antisense strand and a portion of the RNA sequence of interest is preferred. Alternatively, the DsiRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with a portion of the RNA of interest (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or 70.degree. C. hybridization for 12-16 hours; followed by washing). Additional preferred hybridization conditions include hybridization at 70.degree. C. in 1.times.SSC or 50.degree. C. in 1.times.SSC, 50% formamide followed by washing at 70.degree. C. in 0.3.times.SSC or hybridization at 70.degree. C. in 4.times.SSC or 50.degree. C. in 4.times.SSC, 50% formamide followed by washing at 67.degree. C. in 1.times.SSC. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10.degree. C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(.degree. C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(.degree. C.)=81.5+16.6(log 10[Na+])+0.41 (% G+C)-(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1.times.SSC=0.165 M). Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4. The length of the identical nucleotide sequences may be at least about 10, 12, 15, 17, 20, 22, 25, 27 or 30 bases.

[0248] By "conserved sequence region" is meant, a nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system, subject, or organism to another biological system, subject, or organism. The polynucleotide can include both coding and non-coding DNA and RNA.

[0249] By "sense region" is meant a nucleotide sequence of a DsiRNA molecule having complementarity to an antisense region of the DsiRNA molecule. In addition, the sense region of a DsiRNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.

[0250] By "antisense region" is meant a nucleotide sequence of a DsiRNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of a DsiRNA molecule comprises a nucleic acid sequence having complementarity to a sense region of the DsiRNA molecule.

[0251] As used herein, "antisense strand" refers to a single stranded nucleic acid molecule which has a sequence complementary to that of a target RNA. When the antisense strand contains modified nucleotides with base analogs, it is not necessarily complementary over its entire length, but must at least hybridize with a target RNA.

[0252] As used herein, "sense strand" refers to a single stranded nucleic acid molecule which has a sequence complementary to that of an antisense strand. When the antisense strand contains modified nucleotides with base analogs, the sense strand need not be complementary over the entire length of the antisense strand, but must at least duplex with the antisense strand.

[0253] As used herein, "guide strand" refers to a single stranded nucleic acid molecule of a dsRNA or dsRNA-containing molecule, which has a sequence sufficiently complementary to that of a target RNA to result in RNA interference. After cleavage of the dsRNA or dsRNA-containing molecule by Dicer, a fragment of the guide strand remains associated with RISC, binds a target RNA as a component of the RISC complex, and promotes cleavage of a target RNA by RISC. As used herein, the guide strand does not necessarily refer to a continuous single stranded nucleic acid and may comprise a discontinuity, preferably at a site that is cleaved by Dicer. A guide strand is an antisense strand.

[0254] As used herein, "passenger strand" refers to an oligonucleotide strand of a dsRNA or dsRNA-containing molecule, which has a sequence that is complementary to that of the guide strand. As used herein, the passenger strand does not necessarily refer to a continuous single stranded nucleic acid and may comprise a discontinuity, preferably at a site that is cleaved by Dicer. A passenger strand is a sense strand.

[0255] By "target nucleic acid" is meant any nucleic acid sequence whose expression, level or activity is to be modulated. The target nucleic acid can be DNA or RNA. Levels of expression may also be targeted via targeting of upstream effectors of the target of interest, or the effects of a modulated or misregulated target may also be modulated by targeting molecules downstream of, for example, the signaling pathway of a target of interest.

[0256] As is known, RNAi methods are applicable to a wide variety of genes in a wide variety of organisms and the disclosed compositions and methods can be utilized in each of these contexts. Examples of genes which can be targeted by the disclosed compositions and methods include endogenous genes which are genes that are native to the cell or to genes that are not normally native to the cell. Without limitation these genes include oncogenes, cytokine genes, idiotype (Id) protein genes, prion genes, genes that expresses molecules that induce angiogenesis, genes for adhesion molecules, cell surface receptors, proteins involved in metastasis, proteases, apoptosis genes, cell cycle control genes, genes that express EGF and the EGF receptor, multi-drug resistance genes, such as the MDR1 gene.

[0257] More specifically, the target mRNA of the invention specifies the amino acid sequence of a cellular protein (e.g., a nuclear, cytoplasmic, transmembrane, or membrane-associated protein). In another embodiment, the target mRNA of the invention specifies the amino acid sequence of an extracellular protein (e.g., an extracellular matrix protein or secreted protein). As used herein, the phrase "specifies the amino acid sequence" of a protein means that the mRNA sequence is translated into the amino acid sequence according to the rules of the genetic code. The following classes of proteins are listed for illustrative purposes: developmental proteins (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors); oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM I, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor proteins (e.g., BRCA1, BRCA2, MADH4, MCC, NF I, NF2, RBI, TP53, and WTI); and enzymes (e.g., ACC synthases and oxidases, ACP desaturases and hydroxylases, ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases, cellulases, chalcone synthases, chitinases, cyclooxygenases, decarboxylases, dextriinases, DNA and RNA polymerases, galactosidases, glucanases, glucose oxidases, granule-bound starch synthases, GTPases, helicases, hernicellulases, integrases, inulinases, invertases, isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes, nopaline synthases, octopine synthases, pectinesterases, peroxidases, phosphatases, phospholipases, phosphorylases, phytases, plant growth regulator synthases, polygalacturonases, proteinases and peptidases, pullanases, recombinases, reverse transcriptases, RUBISCOs, topoisomerases, and xylanases), ApoB100 and HPRT1.

[0258] In one aspect, the target mRNA molecule of the invention specifies the amino acid sequence of a protein associated with a pathological condition. For example, the protein may be a pathogen-associated protein (e.g., a viral protein involved in immunosuppression of the host, replication of the pathogen, transmission of the pathogen, or maintenance of the infection), or a host protein which facilitates entry of the pathogen into the host, drug metabolism by the pathogen or host, replication or integration of the pathogen's genome, establishment or spread of infection in the host, or assembly of the next generation of pathogen. Pathogens include RNA viruses such as flaviviruses, picornaviruses, rhabdoviruses, filoviruses, retroviruses, including lentiviruses, or DNA viruses such as adenoviruses, poxviruses, herpes viruses, cytomegaloviruses, hepadnaviruses or others. Additional pathogens include bacteria, fungi, helminths, schistosomes and trypanosomes. Other kinds of pathogens can include mammalian transposable elements. Alternatively, the protein may be a tumor-associated protein or an autoimmune disease-associated protein.

[0259] The target gene may be derived from or contained in any organism. The organism may be a plant, animal, protozoa, bacterium, virus or fungus. See e.g., U.S. Pat. No. 6,506,559, incorporated herein by reference.

[0260] In one embodiment of the present invention, each sequence of a DsiRNA molecule of the invention is independently about 25 to about 35 nucleotides in length, in specific embodiments about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length. In another embodiment, the DsiRNA duplexes of the invention independently comprise about 25 to about 30 base pairs (e.g., about 25, 26, 27, 28, 29, or 30). In another embodiment, one or more strands of the DsiRNA molecule of the invention independently comprises about 19 to about 35 nucleotides (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35) that are complementary to a target nucleic acid molecule of interest. Exemplary DsiRNA molecules of the invention are shown in FIG. 1, and below.

[0261] As used herein "cell" is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell. Within certain aspects, the term "cell" refers specifically to mammalian cells, such as human cells, that contain one or more isolated dsNA molecules of the present disclosure. In particular aspects, a cell processes dsRNAs or dsRNA-containing molecules resulting in RNA interference of target nucleic acids, and contains proteins and protein complexes required for RNAi, e.g., Dicer and RISC.

[0262] The DsiRNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through direct dermal application, transdermal application, or injection, with or without their incorporation in biopolymers. In certain aspects of the invention, the dsRNAs of the exemplary structures of dsRNA-peptides presented in FIG. 1 are modified in accordance with the below description of modification patterning of DsiRNA agents. Chemically modified forms of constructs described in FIG. 1, and the below exemplary structures can be used in any and all uses described for the DsiRNA agents described herein.

[0263] In another aspect, the invention provides mammalian cells containing one or more DsiRNA molecules of this invention. The one or more DsiRNA molecules can independently be targeted to the same or different sites.

[0264] By "RNA" is meant a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of a .beta.-D-ribofuranose moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the DsiRNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[0265] By "subject" is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. "Subject" also refers to an organism to which the DsiRNA agents of the invention can be administered. A subject can be a mammal or mammalian cells, including a human or human cells.

[0266] The phrase "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent. Exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. The pharmaceutically acceptable carrier of the disclosed dsRNA compositions may be micellar structures, such as a liposomes, capsids, capsoids, polymeric nanocapsules, or polymeric microcapsules.

[0267] Polymeric nanocapsules or microcapsules facilitate transport and release of the encapsulated or bound dsRNA into the cell. They include polymeric and monomeric materials, especially including polybutylcyanoacrylate. A summary of materials and fabrication methods has been published (see Kreuter, 1991). The polymeric materials which are formed from monomeric and/or oligomeric precursors in the polymerization/nanoparticle generation step, are per se known from the prior art, as are the molecular weights and molecular weight distribution of the polymeric material which a person skilled in the field of manufacturing nanoparticles may suitably select in accordance with the usual skill.

[0268] Various methodologies of the instant invention include step that involves comparing a value, level, feature, characteristic, property, etc. to a "suitable control", referred to interchangeably herein as an "appropriate control". A "suitable control" or "appropriate control" is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing an RNA silencing agent (e.g., DsiRNA) of the invention into a cell or organism. In another embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. In yet another embodiment, a "suitable control" or "appropriate control" is a predefined value, level, feature, characteristic, property, etc.

[0269] The term "in vitro" has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts. The term "in vivo" also has its art recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and/or cells in an organism.

[0270] "Treatment", or "treating" as used herein, is defined as the application or administration of a therapeutic agent (e.g., a DsiRNA agent or a vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disorder with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, or symptoms of the disease or disorder. The term "treatment" or "treating" is also used herein in the context of administering agents prophylactically. The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The term "patient" includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

dsRNA-Peptide Design/Synthesis

[0271] It has been found empirically that longer dsRNA species of from 25 to about 35 nucleotides (DsiRNAs) and especially from 25 to about 30 nucleotides give unexpectedly effective results in terms of potency and duration of action, as compared to 19-23mer siRNA agents. Without wishing to be bound by the underlying theory of the dsRNA processing mechanism, it is thought that the longer dsRNA species serve as a substrate for the Dicer enzyme in the cytoplasm of a cell. In addition to cleaving the dsRNA of the invention into shorter segments, Dicer is thought to facilitate the incorporation of a single-stranded cleavage product derived from the cleaved dsRNA into the RISC complex that is responsible for the destruction of a target RNA of interest. Prior studies (Rossi et al., U.S. Patent Application No. 2007/0265220) have shown that the cleavability of a dsRNA species (specifically, a DsiRNA agent) by Dicer corresponds with increased potency and duration of action of the dsRNA species.

[0272] The invention encompasses dsRNAs comprising double stranded RNAs comprising a first strand and a second strand wherein the first strand and the second strand have a length which is at least 16 and at most 50 nucleotides in length (for example 16-50, 19-35, 19-24, 25-30, 25, 35, 19-23, and 21-23 nucleotides in length).

[0273] A. dsRNAs

[0274] Design of dsRNAs, including DsiRNAs can optionally involve use of predictive scoring algorithms that perform in silico assessments of the projected activity/efficacy of a number of possible DsiRNA agents spanning a region of sequence. Information regarding the design of such scoring algorithms can be found, e.g., in Gong et al. (BMC Bioinformatics 2006, 7:516), though a more recent "v3" algorithm represents a theoretically improved algorithm relative to siRNA scoring algorithms previously available in the art. (The "v3" scoring algorithm is a machine learning algorithm that is not reliant upon any biases in human sequence. In addition, the "v3" algorithm derives from a data set that is approximately three-fold larger than that from which an older "v2" algorithm such as that described in Gong et al. derives.)

[0275] The first and second oligonucleotides of the DsiRNA agents of the instant invention are not required to be completely complementary. In fact, in one embodiment, the 3'-terminus of the sense strand contains one or more mismatches. In one aspect, about two mismatches are incorporated at the 3' terminus of the sense strand. In another embodiment, the DsiRNA of the invention is a double stranded RNA molecule containing two RNA oligonucleotides each of which is 27 nucleotides in length and, when annealed to each other, have blunt ends and a two nucleotide mismatch on the 3'-terminus of the sense strand (the 5'-terminus of the antisense strand). The use of mismatches or decreased thermodynamic stability (specifically at the 3'-sense/5'-antisense position) has been proposed to facilitate or favor entry of the antisense strand into RISC (Schwarz et al., 2003, Cell 115: 199-208; Khvorova et al., 2003, Cell 115: 209-216), presumably by affecting some rate-limiting unwinding steps that occur with entry of the siRNA into RISC. Thus, terminal base composition has been included in design algorithms for selecting active 21mer siRNA duplexes (Ui-Tei et al., 2004, Nucleic Acids Res 32: 936-948; Reynolds et al., 2004, Nat Biotechnol 22: 326-330). With Dicer cleavage of the dsRNA of this embodiment, the small end-terminal sequence which contains the mismatches will either be left unpaired with the antisense strand (become part of a 3'-overhang) or be cleaved entirely off the final 21-mer siRNA. These "mismatches", therefore, do not persist as mismatches in the final RNA component of RISC. The finding that base mismatches or destabilization of segments at the 3'-end of the sense strand of Dicer substrate improved the potency of synthetic duplexes in RNAi, presumably by facilitating processing by Dicer, was a surprising finding of past works describing the design and use of 25-30mer dsRNAs (also termed "DsiRNAs" herein; Rossi et al., U.S. Patent Application Nos. 2005/0277610, 2005/0244858 and 2007/0265220).

[0276] B. Peptides

[0277] The invention provides for compositions comprising a dsRNA of the invention conjugated to a peptide.

[0278] Delivery Peptides

[0279] In certain embodiments the peptide of interest is a delivery peptide as defined hereinabove.

[0280] Delivery Peptide Sequences Useful According to the Invention

[0281] A delivery peptide useful according to the invention increases at least one of onset of action of a dsRNA, duration of action by the delivered dsRNA or speed of delivery of a dsRNA of the invention, as compared to an unconjugated dsRNA. A peptide of the invention decreases, as defined herein, the onset of action such that there is a decrease in the lag time before a dsRNA of interest reaches a target RNA as compared an unconjugated dsRNA. A delivery peptide useful according to the invention increases, as defined herein, the duration of action such that a dsRNA-peptide conjugate inhibits a target RNA for a longer period of time, as compared to an unconjugated dsRNA. A delivery peptide useful according to the invention increases, as defined herein, the speed of delivery of a dsRNA such that a dsRNA-peptide conjugate reaches a target RNA faster than an unconjugated dsRNA.

[0282] According to the invention, an amino acid sequence of a delivery peptide is determined and optimized for the dsRNA to be delivered. Peptide sequences useful for delivery peptides according to the invention are described in the literature.

[0283] In one embodiment, a delivery peptide according to the invention comprises proline residues, for example, a sequence of x1-P-x2-P-x3, where x1 and x3 are any amino acid or peptide segment comprising 2 to 50 amino acids and x2 is either 0 or 1 amino acids or peptide segments containing 2 to 20 amino acids. In another embodiment, x1=a peptide comprising 5 amino acid residues; x2=a peptide comprising 7 amino acid residues and x3=a peptide comprising 4 amino acid residues. In another embodiment, x1=a peptide comprising 8 amino acid residues; x2=a peptide comprising 7 amino acid residues and x3=a peptide comprising 4 amino acid residues. In yet another embodiment, x1=a peptide comprising 8 amino acid residues; x2=a peptide comprising 8 amino acid residues and x3=a peptide comprising 4 amino acid residues (Deber et al., Arch Biochem Biophys. 1986; 251(1):68-76; and Du et al., J Pept Res. 1998; 51(3):235-43.)

[0284] Delivery peptide sequences useful for the invention include, but are not limited to:

TABLE-US-00002 (SEQ ID NO: 1) VRGIITSKTKSLDKGYNKALNDL (SEQ ID NO: 2) VRGIIPFKTKSLDEGYNKALNDL (SEQ ID NO: 3) KSVKAPGI (SEQ ID NO: 4) HKAIDGRSLYNKTLD (SEQ ID NO: 5) LRLTKNSRDDST (SEQ ID NO: 6) KNIVSVKGIRKSI (SEQ ID NO: 7) KSVIPRKGTKAPPRL (SEQ ID NO: 8) KPVMYKNTGKSEQ (SEQ ID NO: 9) EFVMNPANAQGHTPGTRL (SEQ ID NO: 10) EFVMNPANAQGHTAGTRL (SEQ ID NO: 11) EFVMNAANAQGHTPGTRL (SEQ ID NO: 12) EFVMNPANAQGRHTPGTRL (SEQ ID NO: 13) NPKEFVMNPANAQGHTPGTRL (SEQ ID NO: 14) NPKEFVMNPANAQGRHTPGTRL (SEQ ID NO: 15) KKIIPPTNIRENLYNRTASLTDLGGEL (SEQ ID NO: 16) CVRGIITSKTKSLDKGYNKALNDL (SEQ ID NO: 17) CVRGIIPFKTKSLDEGYNKALNDL (SEQ ID NO: 18) CKSVKAPGI (SEQ ID NO: 19) CHKAIDGRSLYNKTLD (SEQ ID NO: 20) CLRLTKNSRDDST (SEQ ID NO: 21) CKNIVSVKGIRKSI (SEQ ID NO: 22) CKSVIPRKGTKAPPRL (SEQ ID NO: 23) CKPVMYKNTGKSEQ (SEQ ID NO: 24) CEFVMNPANAQGHTPGTRL (SEQ ID NO: 25) CEFVMNPANAQGHTAGTRL (SEQ ID NO: 26) CEFVMNAANAQGHTPGTRL (SEQ ID NO: 27) CEFVMNPANAQGRHTPGTRL (SEQ ID NO: 28) CNPKEFVMNPANAQGHTPGTRL (SEQ ID NO: 29) CNPKEFVMNPANAQGRHTPGTRL (SEQ ID NO: 30) CKKIIPPTNIRENLYNRTASLTDLGGEL (SEQ ID NO: 31) GVRGIITSKTKSLDKGYNKALNDL (SEQ ID NO: 32) GVRGIIPFKTKSLDEGYNKALNDL (SEQ ID NO: 33) GKSVKAPGI (SEQ ID NO: 34) GHKAIDGRSLYNKTLD (SEQ ID NO: 35) GLRLTKNSRDDST (SEQ ID NO: 36) GKNIVSVKGIRKSI (SEQ ID NO: 37) GKSVIPRKGTKAPPRL (SEQ ID NO: 38) GKPVMYKNTGKSEQ (SEQ ID NO: 39) GEFVMNPANAQGHTPGTRL (SEQ ID NO: 40) GEFVMNPANAQGHTAGTRL (SEQ ID NO: 41) GEFVMNAANAQGHTPGTRL (SEQ ID NO: 42) GEFVMNPANAQGRHTPGTRL (SEQ ID NO: 43) GNPKEFVMNPANAQGHTPGTRL (SEQ ID NO: 44) GNPKEFVMNPANAQGRHTPGTRL (SEQ ID NO: 45) GKKIIPPTNIRENLYNRTASLTDLGGEL (SEQ ID NO: 46) VRGIITSKTKSLDKGYNKALNDLC (SEQ ID NO: 47) VRGIIPFKTKSLDEGYNKALNDLC (SEQ ID NO: 48) KSVKAPGIC (SEQ ID NO: 49) HKAIDGRSLYNKTLDC (SEQ ID NO: 50) LRLTKNSRDDSTC (SEQ ID NO: 51) KNIVSVKGIRKSIC (SEQ ID NO: 52) KSVIPRKGTKAPPRLC (SEQ ID NO: 53) KPVMYKNTGKSEQC (SEQ ID NO: 54) EFVMNPANAQGHTPGTRLC (SEQ ID NO: 55) EFVMNPANAQGHTAGTRLC (SEQ ID NO: 56) EFVMNAANAQGHTPGTRLC (SEQ ID NO: 57) EFVMNPANAQGRHTPGTRLC (SEQ ID NO: 58) NPKEFVMNPANAQGHTPGTRLC (SEQ ID NO: 59) NPKEFVMNPANAQGRHTPGTRLC (SEQ ID NO: 60) KKIIPPTNIRENLYNRTASLTDLGGELC (SEQ ID NO: 61) KSVKAPGIGGKSVKAPGI (SEQ ID NO: 62) KSVKAPGIGGKSVKAPGIGGKSVKAPGI (SEQ ID NO: 63) KSVKAPGIGG(KSVKAPGI).sub.2 (SEQ ID NO: 64) CKSVKAPGIGGKSVKAPGI (SEQ ID NO: 65) CKSVKAPGIGGKSVKAPGIGGKSVKAPGI (SEQ ID NO: 66) GLFGAIAGFIENGWEGMIDGWYG (SEQ ID NO: 67) CGLFGAIAGFIENGWEGMIDGWYG (SEQ ID NO: 68) GLFGAIAGFIENGWEGMIDGWYGC (SEQ ID NO: 69) GRGDGG (SEQ ID NO: 70) CRGDGG (SEQ ID NO: 71) GRGDGC (SEQ ID NO: 72) THALWHT (SEQ ID NO: 73) GTHALWHT (SEQ ID NO: 74) THALWHTG (SEQ ID NO: 75) CTHALWHT (SEQ ID NO: 76) THALWHTC (SEQ ID NO: 77) QPFMQCLCLIYDASC (SEQ ID NO: 78) GQPFMQCLCLIYDASC (SEQ ID NO: 79) QPFMQCLCLIYDASCG (SEQ ID NO: 80) RNVPPIFNDVYWIAF (SEQ ID NO: 81) GRNVPPIFNDVYWIAF (SEQ ID NO: 82) RNVPPIFNDVYWIAFG (SEQ ID NO: 83) CRNVPPIFNDVYWIAF (SEQ ID NO: 84)

RNVPPIFNDVYWIAFC (SEQ ID NO: 85) VFRVRPWYQSTSQS (SEQ ID NO: 86) GVFRVRPWYQSTSQS (SEQ ID NO: 87) VFRVRPWYQSTSQSG (SEQ ID NO: 88) CVFRVRPWYQSTSQS (SEQ ID NO: 89) VFRVRPWYQSTSQSC (SEQ ID NO: 149) GEFVMNAANAQGHTAGTRL (SEQ ID NO: 150) TQIENLKEKG (SEQ ID NO: 151) H2N-c[D(Cys-Ser-Lys-Cys)]Gly-Peg12-Lys (SEQ ID NO: 152) H2N-c[Cys-Phe-Thr-Lys-D-Trp-Phe-Phe-Cys]-Peg12-Lys (SEQ ID NO: 153) H2N-Thr-Phe-Thr-Lys-D-Trp-Phe-Phe-D-Phe-Peg12-Lys

or portions thereof.

[0285] Targeting Peptides

[0286] In other embodiments, the peptide of interest is a targeting peptide as defined hereinabove.

[0287] According to the invention, an amino acid sequence of a targeting peptide is determined and optimized for the dsRNA that is conjugated to the peptide for delivery. Peptide sequences useful for targeting peptides according to the invention are described in the literature.

[0288] For each ligand family distinct peptide sequence patterns are appropriate. In one embodiment, a peptide useful for targeting the LDL-receptor according to the current invention may contain a sequence of x1-F-x2-YGG-x3, where x1 and x3 are any amino acid or peptide segment containing 2 to 40 amino acids, and x2 is any amino acid. Hussain, Strickland and Bakillah, Annu Rev Nutr. 1999; 19:141-172. Hussain, Front Biosci. 2001; 6:D417-D428.

[0289] Targeting Peptides Useful According to the Invention

[0290] Targeting peptides useful according to the invention include but are not limited to an amino acid sequence from any of the following ligands:

[0291] 1. Parathyroid Hormone (PTH) and PTH-Related Protein

TABLE-US-00003 P01270; PTHY_HUMAN P22858; PTHR_MOUSE Q811S6; Q811S6_MOUSE P01270; PTHY_HUMAN (SEQ ID NO: 90) MIPAKDMAKV MIVMLAICFL TKSDGKSVKK RSVSEIQLMH NLGKHLNSME RVEWLRKKLQ DVHNFVALGA PLAPRDAGSQ RPRKKEDNVL VESHEKSLGE ADKADVNVLT KAKSQ

[0292] 2. Thyroid Stimulating Hormone (TSH)

TABLE-US-00004 P01222; TSHB_HUMAN P12656; TSHB_MOUSE P01222; TSHB_HUMAN (SEQ ID NO: 91) MTALFLMSML FGLACGQAMS FCIPTEYTMH IERRECAYCL TINTTICAGY CMTRDINGKL FLPKYALSQD VCTYRDFIYR TVEIPGCPLH VAPYFSYPVA LSCKCGKCNT DYSDCIHEAI KTNYCTKPQK SYLVGFSV

[0293] 3. TSH Releasing Hormone

TABLE-US-00005 B2R8R1; B2R8R1_HUMAN B2R8R1; B2R8R1_HUMAN (SEQ ID NO: 92) MPGPWLLLAL ALTLNLTGVP GGRAQPEAAQ QEAVTAAEHP GLDDFLRQVE RLLFLRENIQ RLQGDQGEHS ASQIFQSDWL SKRQHPGKRE EEEEEGVEEE EEEEGGAVGP HKRQHPGRRE DEASWSVDVT QHKRQHPGRR SPWLAYAVPK RQHPGRRLAD PKAQRSWEEE EEEEEREEDL MPEKRQHPGK RALGGPCGPQ GAYGQAGLLL GLLDDLSRSQ GAEEKRQHPG RRAAWVREPL EE

[0294] 4. FSH/LH Releasing Hormone

TABLE-US-00006 P01148; GON1_HUMAN O43555; GON2_HUMAN P13562; GON1_MOUSE P01148; GON1_HUMAN (SEQ ID NO: 93) MKPIQKLLAG LILLTWCVEG CSSQHWSYGL RPGGKRDAEN LIDSFQEIVK EVGQLAETQR FECTTHQPRS PLRDLKGALE SLIEEETGQK KI O43555; GON2_HUMAN (SEQ ID NO: 94) MASSRRGLLL LLLLTAHLGP SEAQHWSHGW YPGGKRALSS AQDPQNALRP PGRALDTAAG SPVQTAHGLP SDALAPLDDS MPWEGRTTAQ WSLHRKRHLA RTLLTAAREP RPAPPSSNKV

[0295] 5. Corticotropin Releasing Hormone (CRH)

TABLE-US-00007 P06850; CRF_HUMAN Q8CIT0; CRF_MOUSE P06850; CRF_HUMAN (SEQ ID NO: 95) MRLPLLVSAG VLLVALLPCP PCRALLSRGP VPGARQAPQH PQPLDFFQPP PQSEQPQQPQ ARPVLLRMGE EYFLRLGNLN KSPAAPLSPA SSLLAGGSGS RPSPEQATAN FFRVLLQQLL LPRRSLDSPA ALAERGARNA LGGHQEAPER ERRSEEPPIS LDLTFHLLRE VLEMARAEQL AQQAHSNRKL MEIIGK

[0296] 6. Adrenocorticotropic Hormone (ACTH)

TABLE-US-00008 P01189; COLI_HUMAN P01193; COLI_MOUSE P01189; COLI_HUMAN (SEQ ID NO: 96) MPRSCCSRSG ALLLALLLQA SMEVRGWCLE SSQCQDLTTE SNLLECIRAC KPDLSAETPM FPGNGDEQPL TENPRKYVMG HFRWDRFGRR NSSSSGSSGA GQKREDVSAG EDCGPLPEGG PEPRSDGAKP GPREGKRSYS MEHFRWGKPV GKKRRPVKVY PNGAEDESAE AFPLEFKREL TGQRLREGDG PDGPADDGAG AQADLEHSLL VAAEKKDEGP YRMEHFRWGS PPKDKRYGGF MTSEKSQTPL VTLFKNAIIK NAYKKGE

[0297] 7. Proteinase Activated Receptor (PAR) Ligands and Thrombin Receptor Agonists

TABLE-US-00009 P00734; THRB_HUMAN P19221; THRB_MOUSE P00734; THRB_HUMAN (SEQ ID NO: 97) MAHVRGLQLP GCLALAALCS LVHSQHVFLA PQQARSLLQR VRRANTFLEE VRKGNLEREC VEETCSYEEA FEALESSTAT DVFWAKYTAC ETARTPRDKL AACLEGNCAE GLGTNYRGHV NITRSGIECQ LWRSRYPHKP EINSTTHPGA DLQENFCRNP DSSTTGPWCY TTDPTVRRQE CSIPVCGQDQ VTVAMTPRSE GSSVNLSPPL EQCVPDRGQQ YQGRLAVTTH GLPCLAWASA QAKALSKHQD FNSAVQLVEN FCRNPDGDEE GVWCYVAGKP GDFGYCDLNY CEEAVEEETG DGLDEDSDRA IEGRTATSEY QTFFNPRTFG SGEADCGLRP LFEKKSLEDK TERELLESYI DGRIVEGSDA EIGMSPWQVM LFRKSPQELL CGASLISDRW VLTAAHCLLY PPWDKNFTEN DLLVRIGKHS RTRYERNIEK ISMLEKIYIH PRYNWRENLD RDIALMKLKK PVAFSDYIHP VCLPDRETAA SLLQAGYKGR VTGWGNLKET WTANVGKGQP SVLQVVNLPI VERPVCKDST RIRITDNMFC AGYKPDEGKR GDACEGDSGG PFVMKSPFNN RWYQMGIVSW GEGCDRDGKY GFYTHVFRLK KWIQKVIDQF GE

[0298] 8. Complement Receptor Ligands

TABLE-US-00010 P01024; CO3_HUMAN P01027; CO3_MOUSE P01024; CO3_HUMAN (SEQ ID NO: 98) MGPTSGPSLL LLLLTHLPLA LGSPMYSIIT PNILRLESEE TMVLEAHDAQ GDVPVTVTVH DFPGKKLVLS SEKTVLTPAT NHMGNVTFTI PANREFKSEK GRNKFVTVQA TFGTQVVEKV VLVSLQSGYL FIQTDKTIYT PGSTVLYRIF TVNHKLLPVG RTVMVNIENP EGIPVKQDSL SSQNQLGVLP LSWDIPELVN MGQWKIRAYY ENSPQQVFST EFEVKEYVLP SFEVIVEPTE KFYYIYNEKG LEVTITARFL YGKKVEGTAF VIFGIQDGEQ RISLPESLKR IPIEDGSGEV VLSRKVLLDG VQNPRAEDLV GKSLYVSATV ILHSGSDMVQ AERSGIPIVT SPYQIHFTKT PKYFKPGMPF DLMVFVTNPD GSPAYRVPVA VQGEDTVQSL TQGDGVAKLS INTHPSQKPL SITVRTKKQE LSEAEQATRT MQALPYSTVG NSNNYLHLSV LRTELRPGET LNVNFLLRMD RAHEAKIRYY TYLIMNKGRL LKAGRQVREP GQDLVVLPLS ITTDFIPSFR LVAYYTLIGA SGQREVVADS VWVDVKDSCV GSLVVKSGQS EDRQPVPGQQ MTLKIEGDHG ARVVLVAVDK GVFVLNKKNK LTQSKIWDVV EKADIGCTPG SGKDYAGVFS DAGLTFTSSS GQQTAQRAEL QCPQPAARRR RSVQLTEKRM DKVGKYPKEL RKCCEDGMRE NPMRFSCQRR TRFISLGEAC KKVFLDCCNY ITELRRQHAR ASHLGLARSN LDEDIIAEEN IVSRSEFPES WLWNVEDLKE PPKNGISTKL MNIFLKDSIT TWEILAVSMS DKKGICVADP FEVTVMQDFF IDLRLPYSVV RNEQVEIRAV LYNYRQNQEL KVRVELLHNP AFCSLATTKR RHQQTVTIPP KSSLSVPYVI VPLKTGLQEV EVKAAVYHHF ISDGVRKSLK VVPEGIRMNK TVAVRTLDPE RLGREGVQKE DIPPADLSDQ VPDTESETRI LLQGTPVAQM TEDAVDAERL KHLIVTPSGC GEQNMIGMTP TVIAVHYLDE TEQWEKFGLE KRQGALELIK KGYTQQLAFR QPSSAFAAFV KRAPSTWLTA YVVKVFSLAV NLIAIDSQVL CGAVKWLILE KQKPDGVFQE DAPVIHQEMI GGLRNNNEKD MALTAFVLIS LQEAKDICEE QVNSLPGSIT KAGDFLEANY MNLQRSYTVA IAGYALAQMG RLKGPLLNKF LTTAKDKNRW EDPGKQLYNV EATSYALLAL LQLKDFDFVP PVVRWLNEQR YYGGGYGSTQ ATFMVFQALA QYQKDAPDHQ ELNLDVSLQL PSRSSKITHR IHWESASLLR SEETKENEGF TVTAEGKGQG TLSVVTMYHA KAKDQLTCNK FDLKVTIKPA PETEKRPQDA KNTMILEICT RYRGDQDATM SILDISMMTG FAPDTDDLKQ LANGVDRYIS KYELDKAFSD RNTLIIYLDK VSHSEDDCLA FKVHQYFNVE LIQPGAVKVY AYYNLEESCT RFYHPEKEDG KLNKLCRDEL CRCAEENCFI QKSDDKVTLE ERLDKACEPG VDYVYKTRLV KVQLSNDFDE YIMAIEQTIK SGSDEVQVGQ QRTFISPIKC REALKLEEKK HYLMWGLSSD FWGEKPNLSY IIGKDTWVEH WPEEDECQDE ENQKQCQDLG AFTESMVVFG CPN P0C0L5; CO4B_HUMAN (SEQ ID NO: 99) MRLLWGLIWA SSFFTLSLQK PRLLLFSPSV VHLGVPLSVG VQLQDVPRGQ VVKGSVFLRN PSRNNVPCSP KVDFTLSSER DFALLSLQVP LKDAKSCGLH QLLRGPEVQL VAHSPWLKDS LSRTTNIQGI NLLFSSRRGH LFLQTDQPIY NPGQRVRYRV FALDQKMRPS TDTITVMVEN SHGLRVRKKE VYMPSSIFQD DFVIPDISEP GTWKISARFS DGLESNSSTQ FEVKKYVLPN FEVKITPGKP YILTVPGHLD EMQLDIQARY IYGKPVQGVA YVRFGLLDED GKKTFFRGLE SQTKLVNGQS HISLSKAEFQ DALEKLNMGI TDLQGLRLYV AAAIIESPGG EMEEAELTSW YFVSSPFSLD LSKTKRHLVP GAPFLLQALV REMSGSPASG IPVKVSATVS SPGSVPEVQD IQQNTDGSGQ VSIPIIIPQT ISELQLSVSA GSPHPAIARL TVAAPPSGGP GFLSIERPDS RPPRVGDTLN LNLRAVGSGA TFSHYYYMIL SRGQIVFMNR EPKRTLTSVS VFVDHHLAPS FYFVAFYYHG DHPVANSLRV DVQAGACEGK LELSVDGAKQ YRNGESVKLH LETDSLALVA LGALDTALYA AGSKSHKPLN MGKVFEAMNS YDLGCGPGGG DSALQVFQAA GLAFSDGDQW TLSRKRLSCP KEKTTRKKRN VNFQKAINEK LGQYASPTAK RCCQDGVTRL PMMRSCEQRA ARVQQPDCRE PFLSCCQFAE SLRKKSRDKG QAGLQRALEI LQEEDLIDED DIPVRSFFPE NWLWRVETVD RFQILTLWLP DSLTTWEIHG LSLSKTKGLC VATPVQLRVF REFHLHLRLP MSVRRFEQLE LRPVLYNYLD KNLTVSVHVS PVEGLCLAGG GGLAQQVLVP AGSARPVAFS VVPTAAAAVS LKVVARGSFE FPVGDAVSKV LQIEKEGAIH REELVYELNP LDHRGRTLEI PGNSDPNMIP DGDFNSYVRV TASDPLDTLG SEGALSPGGV ASLLRLPRGC GEQTMIYLAP TLAASRYLDK TEQWSTLPPE TKDHAVDLIQ KGYMRIQQFR KADGSYAAWL SRDSSTWLTA FVLKVLSLAQ EQVGGSPEKL QETSNWLLSQ QQADGSFQDL SPVIHRSMQG GLVGNDETVA LTAFVTIALH HGLAVFQDEG AEPLKQRVEA SISKANSFLG EKASAGLLGA HAAAITAYAL SLTKAPVDLL GVAHNNLMAM AQETGDNLYW GSVTGSQSNA VSPTPAPRNP SDPMPQAPAL WIETTAYALL HLLLHEGKAE MADQASAWLT RQGSFQGGFR STQDTVIALD ALSAYWIASH TTEERGLNVT LSSTGRNGFK SHALQLNNRQ IRGLEEELQF SLGSKINVKV GGNSKGTLKV LRTYNVLDMK NTTCQDLQIE VTVKGHVEYT MEANEDYEDY EYDELPAKDD PDAPLQPVTP LQLFEGRRNR RRREAPKVVE EQESRVHYTV CIWRNGKVGL SGMAIADVTL LSGFHALRAD LEKLTSLSDR YVSHFETEGP HVLLYFDSVP TSRECVGFEA VQEVPVGLVQ PASATLYDYY NPERRCSVFY GAPSKSRLLA TLCSAEVCQC AEGKCPRQRR ALERGLQDED GYRMKFACYY PRVEYGFQVK VLREDSRAAF RLFETKITQV LHFTKDVKAA ANQMRNFLVR ASCRLRLEPG KEYLIMGLDG ATYDLEGHPQ YLLDSNSWIE EMPSERLCRS TRQRAACAQL NDFLQEYGTQ GCQV

[0299] 9. Ligands for LDL Receptor Family

TABLE-US-00011 P05067; A4_HUMAN P12023; A4_MOUSE P05067; A4_HUMAN (SEQ ID NO: 100) MLPGLALLLL AAWTARALEV PTDGNAGLLA EPQIAMFCGR LNMHMNVQNG KWDSDPSGTK TCIDTKEGIL QYCQEVYPEL QITNVVEANQ PVTIQNWCKR GRKQCKTHPH FVIPYRCLVG EFVSDALLVP DKCKFLHQER MDVCETHLHW HTVAKETCSE KSTNLHDYGM LLPCGIDKFR GVEFVCCPLA EESDNVDSAD AEEDDSDVWW GGADTDYADG SEDKVVEVAE EEEVAEVEEE EADDDEDDED GDEVEEEAEE PYEEATERTT SIATTTTTTT ESVEEVVREV CSEQAETGPC RAMISRWYFD VTEGKCAPFF YGGCGGNRNN FDTEEYCMAV CGSAMSQSLL KTTQEPLARD PVKLPTTAAS TPDAVDKYLE TPGDENEHAH FQKAKERLEA KHRERMSQVM REWEEAERQA KNLPKADKKA VIQHFQEKVE SLEQEAANER QQLVETHMAR VEAMLNDRRR LALENYITAL QAVPPRPRHV FNMLKKYVRA EQKDRQHTLK HFEHVRMVDP KKAAQIRSQV MTHLRVIYER MNQSLSLLYN VPAVAEEIQD EVDELLQKEQ NYSDDVLANM ISEPRISYGN DALMPSLTET KTTVELLPVN GEFSLDDLQP WHSFGADSVP ANTENEVEPV DARPAADRGL TTRPGSGLTN IKTEEISEVK MDAEFRHDSG YEVHHQKLVF FAEDVGSNKG AIIGLMVGGV VIATVIVITL VMLKKKQYTS IHHGVVEVDA AVTPEERHLS KMQQNGYENP TYKFFEQMQN

[0300] 10. Endocrine and Exocrine Receptor Ligands

TABLE-US-00012 P01241; SOMA_HUMAN P06880; SOMA_MOUSE Q9UBU3; GHRL_HUMAN Q9EQX0; GHRL_MOUSE P01241; SOMA_HUMAN (SEQ ID NO: 101) MATGSRTSLL LAFGLLCLPW LQEGSAFPTI PLSRLFDNAM LRAHRLHQLA FDTYQEFEEA YIPKEQKYSF LQNPQTSLCF SESIPTPSNR EETQQKSNLE LLRISLLLIQ SWLEPVQFLR SVFANSLVYG ASDSNVYDLL KDLEEGIQTL MGRLEDGSPR TGQIFKQTYS KFDTNSHNDD ALLKNYGLLY CFRKDMDKVE TFLRIVQCRS VEGSCGF Q9UBU3; GHRL_HUMAN (SEQ ID NO: 102) MPSPGTVCSL LLLGMLWLDL AMAGSSFLSP EHQRVQQRKE SKKPPAKLQP RALAGWLRPE DGGQAEGAED ELEVRFNAPF DVGIKLSGVQ YQQHSQALGK FLQDILWEEA KEAPADK

[0301] 11. Transforming Growth Factor Ligands

TABLE-US-00013 P01137; TGFB1_HUMAN P04202; TGFB1_MOUSE P01137; TGFB1_HUMAN (SEQ ID NO: 103) MPPSGLRLLL LLLPLLWLLV LTPGRPAAGL STCKTIDMEL VKRKRIEAIR GQILSKLRLA SPPSQGEVPP GPLPEAVLAL YNSTRDRVAG ESAEPEPEPE ADYYAKEVTR VLMVETHNEI YDKFKQSTHS IYMFFNTSEL REAVPEPVLL SRAELRLLRL KLKVEQHVEL YQKYSNNSWR YLSNRLLAPS DSPEWLSFDV TGVVRQWLSR GGEIEGFRLS AHCSCDSRDN TLQVDINGFT TGRRGDLATI HGMNRPFLLL MATPLERAQH LQSSRHRRAL DTNYCFSSTE KNCCVRQLYI DFRKDLGWKW IHEPKGYHAN FCLGPCPYIW SLDTQYSKVL ALYNQHNPGA SAAPCCVPQA LEPLPIVYYV GRKPKVEQLS NMIVRSCKCS

[0302] 12. Chemokine Receptor Ligands

TABLE-US-00014 P13500; CCL2_HUMAN P10148; CCL2_MOUSE P13500; CCL2_HUMAN (SEQ ID NO: 104) MKVSAALLCL LLIAATFIPQ GLAQPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

[0303] 13. Integrins

TABLE-US-00015 P05556; ITB1_HUMAN P09055; ITB1_MOUSE P05556; ITB1_HUMAN (SEQ ID NO: 105) MNLQPIFWIG LISSVCCVFA QTDENRCLKA NAKSCGECIQ AGPNCGWCTN STFLQEGMPT SARCDDLEAL KKKGCPPDDI ENPRGSKDIK KNKNVTNRSK GTAEKLKPED ITQIQPQQLV LRLRSGEPQT FTLKFKRAED YPIDLYYLMD LSYSMKDDLE NVKSLGTDLM NEMRRITSDF RIGFGSFVEK TVMPYISTTP AKLRNPCTSE QNCTSPFSYK NVLSLTNKGE VFNELVGKQR ISGNLDSPEG GFDAIMQVAV CGSLIGWRNV TRLLVFSTDA GFHFAGDGKL GGIVLPNDGQ CHLENNMYTM SHYYDYPSIA HLVQKLSENN IQTIFAVTEE FQPVYKELKN LIPKSAVGTL SANSSNVIQL IIDAYNSLSS EVILENGKLS EGVTISYKSY CKNGVNGTGE NGRKCSNISI GDEVQFEISI TSNKCPKKDS DSFKIRPLGF TEEVEVILQY ICECECQSEG IPESPKCHEG NGTFECGACR CNEGRVGRHC ECSTDEVNSE DMDAYCRKEN SSEICSNNGE CVCGQCVCRK RDNTNEIYSG KFCECDNFNC DRSNGLICGG NGVCKCRVCE CNPNYTGSAC DCSLDTSTCE ASNGQICNGR GICECGVCKC TDPKFQGQTC EMCQTCLGVC AEHKECVQCR AFNKGEKKDT CTQECSYFNI TKVESRDKLP QPVQPDPVSH CKEKDVDDCW FYFTYSVNGN NEVMVHVVEN PECPTGPDII PIVAGVVAGI VLIGLALLLI WKLLMIIHDR REFAKFEKEK MNAKWDTGEN PIYKSAVTTV VNPKYEGK

[0304] 14. Interleukins

TABLE-US-00016 Q13169; Q13169_HUMAN Q0PGS4; Q0PGS4_MOUSE Q13169; Q13169_HUMAN (SEQ ID NO: 106) MYRMQLLSCI ALILALVTNS APTSSSTKKT KKTQLQLEHL LLDLQMILNG INNYKNPKLT RMLTFKFYMP KKATELKQLQ CLEEELKPLE EVLNLAQSKN FHLRPRDLIS NINVIVLELK GSETTFMCEY ADETATIVEF LNRWITFCQS IISTLT

[0305] 15. Differentiation Factors Like Bone Differentiation Factors

TABLE-US-00017 P13497; BMP1_HUMAN P98063; BMP1_MOUSE P13497; BMP1_HUMAN (SEQ ID NO: 107) MPGVARLPLL LGLLLLPRPG RPLDLADYTY DLAEEDDSEP LNYKDPCKAA AFLGDIALDE EDLRAFQVQQ AVDLRRHTAR KSSIKAAVPG NTSTPSCQST NGQPQRGACG RWRGRSRSRR AATSRPERVW PDGVIPFVIG GNFTGSQRAV FRQAMRHWEK HTCVTFLERT DEDSYIVFTY RPCGCCSYVG RRGGGPQAIS IGKNCDKFGI VVHELGHVVG FWHEHTRPDR DRHVSIVREN IQPGQEYNFL KMEPQEVESL GETYDFDSIM HYARNTFSRG IFLDTIVPKY EVNGVKPPIG QRTRLSKGDI AQARKLYKCP ACGETLQDST GNFSSPEYPN GYSAHMHCVW RISVTPGEKI ILNFTSLDLY RSRLCWYDYV EVRDGFWRKA PLRGRFCGSK LPEPIVSTDS RLWVEFRSSS NWVGKGFFAV YEAICGGDVK KDYGHIQSPN YPDDYRPSKV CIWRIQVSEG FHVGLTFQSF EIERHDSCAY DYLEVRDGHS ESSTLIGRYC GYEKPDDIKS TSSRLWLKFV SDGSINKAGF AVNFFKEVDE CSRPNRGGCE QRCLNTLGSY KCSCDPGYEL APDKRRCEAA CGGFLTKLNG SITSPGWPKE YPPNKNCIWQ LVAPTQYRIS LQFDFFETEG NDVCKYDFVE VRSGLTADSK LHGKFCGSEK PEVITSQYNN MRVEFKSDNT VSKKGFKAHF FSDKDECSKD NGGCQQDCVN TFGSYECQCR SGFVLHDNKH DCKEAGCDHK VTSTSGTITS PNWPDKYPSK KECTWAISST PGHRVKLTFM EMDIESQPEC AYDHLEVFDG RDAKAPVLGR FCGSKKPEPV LATGSRMFLR FYSDNSVQRK GFQASHATEC GGQVRADVKT KDLYSHAQFG DNNYPGGVDC EWVIVAEEGY GVELVFQTFE VEEETDCGYD YMELFDGYDS TAPRLGRYCG SGPPEEVYSA GDSVLVKFHS DDTITKKGFH LRYTSTKFQD TLHSRK

[0306] 16. Gastrin-Releasing Peptide

TABLE-US-00018 P07492; GRP_HUMAN Q8R1I2; GRP_MOUSE P07492; GRP_HUMAN (SEQ ID NO: 108) MRGSELPLVL LALVLCLAPR GRAVPLPAGG GTVLTKMYPR GNHWAVGHLM GKKSTGESSS VSERGSLKQQ LREYIRWEEA ARNLLGLIEA KENRNHQPPQ PKALGNQQPS WDSEDSSNFK DVGSKGKVGR LSAPGSQREG RNPQLNQQ

[0307] 17. Vasoactive Intestinal Peptide (VIP)

TABLE-US-00019 P01282; VIP_HUMAN P32648; VIP_MOUSE P01282; VIP_HUMAN (SEQ ID NO: 109) MDTRNKAQLL VLLTLLSVLF SQTSAWPLYR APSALRLGDR IPFEGANEPD QVSLKEDIDM LQNALAENDT PYYDVSRNAR HADGVFTSDF SKLLGQLSAK KYLESLMGKR VSSNISEDPV PVKRHSDAVF TDNYTRLRKQ MAVKKYLNSI LNGKRSSEGE SPDFPEELEK

[0308] 18. Insulin and Insulin-Like Growth Factor

TABLE-US-00020 P01308; INS_HUMAN P01343; IGF1A_HUMAN P05019; IGF1B_HUMAN P05017; IGF1_MOUSE P01308; INS_HUMAN (SEQ ID NO: 110) MALWMRLLPL LALLALWGPD PAAAFVNQHL CGSHLVEALY LVCGERGFFY TPKTRREAED LQVGQVELGG GPGAGSLQPL ALEGSLQKRG IVEQCCTSIC SLYQLENYCN P01343; IGF1A_HUMAN (SEQ ID NO: 111) MGKISSLPTQ LFKCCFCDFL KVKMHTMSSS HLFYLALCLL TFTSSATAGP ETLCGAELVD ALQFVCGDRG FYFNKPTGYG SSSRRAPQTG IVDECCFRSC DLRRLEMYCA PLKPAKSARS VRAQRHTDMP KTQKEVHLKN ASRGSAGNKN YRM P05019; IGF1B_HUMAN (SEQ ID NO: 112) MGKISSLPTQ LFKCCFCDFL KVKMHTMSSS HLFYLALCLL TFTSSATAGP ETLCGAELVD ALQFVCGDRG FYFNKPTGYG SSSRRAPQTG IVDECCFRSC DLRRLEMYCA PLKPAKSARS VRAQRHTDMP KTQKYQPPST NKNTKSQRRK GWPKTHPGGE QKEGTEASLQ IRGKKKEQRR EIGSRNAECR GKKGK

[0309] 19. Calcitonin and Calcitonin Gene-Related Peptide

TABLE-US-00021 P01258; CALC_HUMAN P70160; CALC_MOUSE P01258; CALC_HUMAN (SEQ ID NO: 113) MGFQKFSPFL ALSILVLLQA GSLHAAPFRS ALESSPADPA TLSEDEARLL LAALVQDYVQ MKASELEQEQ EREGSSLDSP RSKRCGNLST CMLGTYTQDF NKFHTFPQTA IGVGAPGKKR DMSSDLERDH RPHVSMPQNA N

[0310] 20. Ligands to inflammatory cells like mast cells, eosinophils, macrophage, Monocytes, and Neutrophils

TABLE-US-00022 P09603; CSF1_HUMAN P07141; CSF1_MOUSE P0C0L5; CO4B_HUMAN P01029; CO4B_MOUSE P09603; CSF1_HUMAN (SEQ ID NO: 114) MTAPGAAGRC PPTTWLGSLL LLVCLLASRS ITEEVSEYCS HMIGSGHLQS LQRLIDSQME TSCQITFEFV DQEQLKDPVC YLKKAFLLVQ DIMEDTMRFR DNTPNAIAIV QLQELSLRLK SCFTKDYEEH DKACVRTFYE TPLQLLEKVK NVFNETKNLL DKDWNIFSKN CNNSFAECSS QDVVTKPDCN CLYPKAIPSS DPASVSPHQP LAPSMAPVAG LTWEDSEGTE GSSLLPGEQP LHTVDPGSAK QRPPRSTCQS FEPPETPVVK DSTIGGSPQP RPSVGAFNPG MEDILDSAMG TNWVPEEASG EASEIPVPQG TELSPSRPGG GSMQTEPARP SNFLSASSPL PASAKGQQPA DVTGTALPRV GPVRPTGQDW NHTPQKTDHP SALLRDPPEP GSPRISSLRP QGLSNPSTLS AQPQLSRSHS SGSVLPLGEL EGRRSTRDRR SPAEPEGGPA SEGAARPLPR FNSVPLTDTG HERQSEGSSS PQLQESVFHL LVPSVILVLL AVGGLLFYRW RRRSHQEPQR ADSPLEQPEG SPLTQDDRQV ELPV

[0311] Additional targeting peptides useful according to the invention include but are not limited to the following:

TABLE-US-00023 GTFVYGGCRAKRNNFKSAED (SEQ ID NO: 115) GPFFYGGCGGNRNNFDTEEY (SEQ ID NO: 116) GTFFYGGCRGKRNNFKTEEY (SEQ ID NO: 117) GTFFYGGSRGKRNNFKTEEY (SEQ ID NO: 118) GRFFYGGSRGKRNNFRTEEY (SEQ ID NO: 119) GTFFYGGSRGRRNNFRTEEY (SEQ ID NO: 120) CTFVYGGCRAKRNNFKSAED (SEQ ID NO: 121) CPFFYGGCGGNRNNFDTEEY (SEQ ID NO: 122) CTFFYGGCRGKRNNFKTEEY (SEQ ID NO: 123) CTFFYGGSRGKRNNFKTEEY (SEQ ID NO: 124) CRFFYGGSRGKRNNFRTEEY (SEQ ID NO: 125) CTFFYGGSRGRRNNFRTEEY (SEQ ID NO: 126) TFVYGGCRAKRNNFKSAEDG (SEQ ID NO: 127) PFFYGGCGGNRNNFDTEEYG (SEQ ID NO: 128) TFFYGGCRGKRNNFKTEEYG (SEQ ID NO: 129) TFFYGGSRGKRNNFKTEEYG (SEQ ID NO: 130) RFFYGGSRGKRNNFRTEEYG (SEQ ID NO: 131) TFFYGGSRGRRNNFRTEEYG (SEQ ID NO: 132) TFVYGGCRAKRNNFKSAEDC (SEQ ID NO: 133) PFFYGGCGGNRNNFDTEEYC (SEQ ID NO: 134) TFFYGGCRGKRNNFKTEEYC (SEQ ID NO: 135) TFFYGGSRGKRNNFKTEEYC (SEQ ID NO: 136) RFFYGGSRGKRNNFRTEEYC (SEQ ID NO: 137) TFFYGGSRGRRNNFRTEEYC (SEQ ID NO: 138)

[0312] In one embodiment, a peptide of the invention is conjugated to a translocation domain, for example a translocation domain of a neurotoxin. Neurotoxin translocation domain peptide sequences that are useful according to the invention include but are not limited the following. Peptides sequences are chosen from any subunit within the sequence. Peptide segments based on the sequences that meet the specifications of the invention are chosen.

[0313] 1. Botulinum Neurotoxin Type A (BoNT/A) (EC 3.4.24.69) (Bontoxilysin-A)

TABLE-US-00024 P10845; BXA1_CLOBO (SEQ ID NO: 139) MPFVNKQFNY KDPVNGVDIA YIKIPNVGQM QPVKAFKIHN KIWVIPERDT FTNPEEGDLN PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VTKLFERIYS TDLGRMLLTS IVRGIPFWGG STIDTELKVI DTNCINVIQP DGSYRSEELN LVIIGPSADI IQFECKSFGH EVLNLTRNGY GSTQYIRFSP DFTFGFEESL EVDTNPLLGA GKFATDPAVT LAHELIHAGH RLYGIAINPN RVFKVNTNAY YEMSGLEVSF EELRTFGGHD AKFIDSLQEN EFRLYYYNKF KDIASTLNKA KSIVGTTASL QYMKNVFKEK YLLSEDTSGK FSVDKLKFDK LYKMLTEIYT EDNFVKFFKV LNRKTYLNFD KAVFKINIVP KVNYTIYDGF NLRNTNLAAN FNGQNTEINN MNFTKLKNFT GLFEFYKLLC VRGIITSKTK SLDKGYNKAL NDLCIKVNNW DLFFSPSEDN FTNDLNKGEE ITSDTNIEAA EENISLDLIQ QYYLTFNFDN EPENISIENL SSDIIGQLEL MPNIERFPNG KKYELDKYTM FHYLRAQEFE HGKSRIALTN SVNEALLNPS RVYTFFSSDY VKKVNKATEA AMFLGWVEQL VYDFTDETSE VSTTDKIADI TIIIPYIGPA LNIGNMLYKD DFVGALIFSG AVILLEFIPE IAIPVLGTFA LVSYIANKVL TVQTIDNALS KRNEKWDEVY KYIVTNWLAK VNTQIDLIRK KMKEALENQA EATKAIINYQ YNQYTEEEKN NINFNIDDLS SKLNESINKA MININKFLNQ CSVSYLMNSM IPYGVKRLED FDASLKDALL KYIYDNRGTL IGQVDRLKDK VNNTLSTDIP FQLSKYVDNQ RLLSTFTEYI KNIINTSILN LRYESNHLID LSRYASKINI GSKVNFDPID KNQIQLFNLE SSKIEVILKN AIVYNSMYEN FSTSFWIRIP KYFNSISLNN EYTIINCMEN NSGWKVSLNY GEIIWTLQDT QEIKQRVVFK YSQMINISDY INRWIFVTIT NNRLNNSKIY INGRLIDQKP ISNLGNIHAS NNIMFKLDGC RDTHRYIWIK YFNLFDKELN EKEIKDLYDN QSNSGILKDF WGDYLQYDKP YYMLNLYDPN KYVDVNNVGI RGYMYLKGPR GSVMTTNIYL NSSLYRGTKF IIKKYASGNK DNIVRNNDRV YINVVVKNKE YRLATNASQA GVEKILSALE IPDVGNLSQV VVMKSKNDQG ITNKCKMNLQ DNNGNDIGFI GFHQFNNIAK LVASNWYNRQ IERSSRTLGC SWEFIPVDDG WGERPL

[0314] 2. Botulinum Neurotoxin Type B (BoNT/B) (EC 3.4.24.69) (Bontoxilysin-B)

TABLE-US-00025 B1INP5; BXB_CLOBK (SEQ ID NO: 140) MPVTINNFNY NDPIDNNNII MMEPPFARGT GRYYKAFKIT DRIWIIPERY TFGYKPEDFN KSSGIFNRDV CEYYDPDYLN TNDKKNIFLQ TMIKLFNRIK SKPLGEKLLE MIINGIPYLG DRRVPLEEFN TNIASVTVNK LISNPGEVER KKGIFANLII FGPGPVLNEN ETIDIGIQNH FASREGFGGI MQMKFCPEYV SVFNNVQENK GASIFNRRGY FSDPALILMH ELIHVLHGLY GIKVDDLPIV PNEKKFFMQS TDAIQAEELY TFGGQDPSII TPSTDKSIYD KVLQNFRGIV DRLNKVLVCI SDPNININIY KNKFKDKYKF VEDSEGKYSI DVESFDKLYK SLMFGFTETN IAENYKIKTR ASYFSDSLPP VKIKNLLDNE IYTIEEGFNI SDKDMEKEYR GQNKAINKQA YEEISKEHLA VYKIQMCKSV KAPGICIDVD NEDLFFIADK NSFSDDLSKN ERIEYNTQSN YIENDFPINE LILDTDLISK IELPSENTES LTDFNVDVPV YEKQPAIKKI FTDENTIFQY LYSQTFPLDI RDISLTSSFD DALLFSNKVY SFFSMDYIKT ANKVVEAGLF AGWVKQIVND FVIEANKSNT MDKIADISLI VPYIGLALNV GNETAKGNFE NAFEIAGASI LLEFIPELLI PVVGAFLLES YIDNKNKIIK TIDNALTKRN EKWSDMYGLI VAQWLSTVNT QFYTIKEGMY KALNYQAQAL EEIIKYRYNI YSEKEKSNIN IDFNDINSKL NEGINQAIDN INNFINGCSV SYLMKKMIPL AVEKLLDFDN TLKKNLLNYI DENKLYLIGS AEYEKSKVNK YLKTIMPFDL SIYTNDTILI EMFNKYNSEI LNNIILNLRY KDNNLIDLSG YGAKVEVYDG VELNDKNQFK LTSSANSKIR VTQNQNIIFN SVFLDFSVSF WIRIPKYKND GIQNYIHNEY TIINCMKNNS GWKISIRGNR IIWTLIDING KTKSVFFEYN IREDISEYIN RWFFVTITNN LNNAKIYING KLESNTDIKD IREVIANGEI IFKLDGDIDR TQFIWMKYFS IFNTELSQSN IEERYKIQSY SEYLKDFWGN PLMYNKEYYM FNAGNKNSYI KLKKDSPVGE ILTRSKYNQN SKYINYRDLY IGEKFIIRRK SNSQSINDDI VRKEDYIYLD FFNLNQEWRV YTYKYFKKEE EKLFLAPISD SDEFYNTIQI KEYDEQPTYS CQLLFKKDEE STDEIGLIGI HRFYESGIVF EEYKDYFCIS KWYLKEVKRK PYNLKLGCNW QFIPKDEGWT E

[0315] 3. Botulinum Neurotoxin Type C1 (BoNT/C1) (EC 3.4.24.69) (Bontoxilysin-C1)

TABLE-US-00026 P18640; BXC1_CLOBO (SEQ ID NO: 141) MPITINNFNY SDPVDNKNIL YLDTHLNTLA NEPEKAFRIT GNIWVIPDRF SRNSNPNLNK PPRVTSPKSG YYDPNYLSTD SDKDPFLKEI IKLFKRINSR EIGEELIYRL STDIPFPGNN NTPINTFDFD VDFNSVDVKT RQGNNWVKTG SINPSVIITG PRENIIDPET STFKLTNNTF AAQEGFGALS IISISPRFML TYSNATNDVG EGRFSKSEFC MDPILILMHE LNHAMHNLYG IAIPNDQTIS SVTSNIFYSQ YNVKLEYAEI YAFGGPTIDL IPKSARKYFE EKALDYYRSI AKRLNSITTA NPSSFNKYIG EYKQKLIRKY RFVVESSGEV TVNRNKFVEL YNELTQIFTE FNYAKIYNVQ NRKIYLSNVY TPVTANILDD NVYDIQNGFN IPKSNLNVLF MGQNLSRNPA LRKVNPENML YLFTKFCHKA IDGRSLYNKT LDCRELLVKN TDLPFIGDIS DVKTDIFLRK DINEETEVIY YPDNVSVDQV ILSKNTSEHG QLDLLYPSID SESEILPGEN QVFYDNRTQN VDYLNSYYYL ESQKLSDNVE DFTFTRSIEE ALDNSAKVYT YFPTLANKVN AGVQGGLFLM WANDVVEDFT TNILRKDTLD KISDVSAIIP YIGPALNISN SVRRGNFTEA FAVTGVTILL EAFPEFTIPA LGAFVIYSKV QERNEIIKTI DNCLEQRIKR WKDSYEWMMG TWLSRIITQF NNISYQMYDS LNYQAGAIKA KIDLEYKKYS GSDKENIKSQ VENLKNSLDV KISEAMNNIN KFIRECSVTY LFKNMLPKVI DELNEFDRNT KAKLINLIDS HNIILVGEVD KLKAKVNNSF QNTIPFNIFS YTNNSLLKDI INEYFNNIND SKILSLQNRK NTLVDTSGYN AEVSEEGDVQ LNPIFPFDFK LGSSGEDRGK VIVTQNENIV YNSMYESFSI SFWIRINKWV SNLPGYTIID SVKNNSGWSI GIISNFLVFT LKQNEDSEQS INFSYDISNN APGYNKWFFV TVTNNMMGNM KIYINGKLID TIKVKELTGI NFSKTITFEI NKIPDTGLIT SDSDNINMWI RDFYIFAKEL DGKDINILFN SLQYTNVVKD YWGNDLRYNK EYYMVNIDYL NRYMYANSRQ IVFNTRRNNN DFNEGYKIII KRIRGNTNDT RVRGGDILYF DMTINNKAYN LFMKNETMYA DNHSTEDIYA IGLREQTKDI NDNIIFQIQP MNNTYYYASQ IFKSNFNGEN ISGICSIGTY RFRLGGDWYR HNYLVPTVKQ GNYASLLEST STHWGFVPVS E

[0316] 4. Botulinum Neurotoxin Type D (BoNT/D) (EC 3.4.24.69) (Bontoxilysin-D)

TABLE-US-00027 P19321; BXD_CLOBO (SEQ ID NO: 142) MTWPVKDFNY SDPVNDNDIL YLRIPQNKLI TTPVKAFMIT QNIWVIPERF SSDTNPSLSK PPRPTSKYQS YYDPSYLSTD EQKDTFLKGI IKLFKRINER DIGKKLINYL VVGSPFMGDS STPEDTFDFT RHTTNIAVEK FENGSWKVTN IITPSVLIFG PLPNILDYTA SLTLQGQQSN PSFEGFGTLS ILKVAPEFLL TFSDVTSNQS SAVLGKSIFC MDPVIALMHE LTHSLHQLYG INIPSDKRIR PQVSEGFFSQ DGPNVQFEEL YTFGGLDVEI IPQIERSQLR EKALGHYKDI AKRLNNINKT IPSSWISNID KYKKIFSEKY NFDKDNTGNF VVNIDKFNSL YSDLTNVMSE VVYSSQYNVK NRTHYFSRHY LPVFANILDD NIYTIRDGFN LTNKGFNIEN SGQNIERNPA LQKLSSESVV DLFTKVCLRL TKNSRDDSTC IKVKNNRLPY VADKDSISQE IFENKIITDE TNVQNYSDKF SLDESILDGQ VPINPEIVDP LLPNVNMEPL NLPGEEIVFY DDITKYVDYL NSYYYLESQK LSNNVENITL TTSVEEALGY SNKIYTFLPS LAEKVNKGVQ AGLFLNWANE VVEDFTTNIM KKDTLDKISD VSVIIPYIGP ALNIGNSALR GNFNQAFATA GVAFLLEGFP EFTIPALGVF TFYSSIQERE KIIKTIENCL EQRVKRWKDS YQWMVSNWLS RITTQFNHIN YQMYDSLSYQ ADAIKAKIDL EYKKYSGSDK ENIKSQVENL KNSLDVKISE AMNNINKFIR ECSVTYLFKN MLPKVIDELN KFDLRTKTEL INLIDSHNII LVGEVDRLKA KVNESFENTM PFNIFSYTNN SLLKDIINEY FNSINDSKIL SLQNKKNALV DTSGYNAEVR VGDNVQLNTI YTNDFKLSSS GDKIIVNLNN NILYSAIYEN SSVSFWIKIS KDLTNSHNEY TIINSIEQNS GWKLCIRNGN IEWILQDVNR KYKSLIFDYS ESLSHTGYTN KWFFVTITNN IMGYMKLYIN GELKQSQKIE DLDEVKLDKT IVFGIDENID ENQMLWIRDF NIFSKELSNE DINIVYEGQI LRNVIKDYWG NPLKFDTEYY IINDNYIDRY IAPESNVLVL VQYPDRSKLY TGNPITIKSV SDKNPYSRIL NGDNIILHML YNSRKYMIIR DTDTIYATQG GECSQNCVYA LKLQSNLGNY GIGIFSIKNI VSKNKYCSQI FSSFRENTML LADIYKPWRF SFKNAYTPVA VTNYETKLLS TSSFWKFISR DPGWVE

[0317] 5. Botulinum Neurotoxin Type E (BoNT/E) (EC 3.4.24.69) (Bontoxilysin-E)

TABLE-US-00028 Q00496; BXE_CLOBO (SEQ ID NO: 143) MPKINSFNYN DPVNDRTILY IKPGGCQEFY KSFNIMKNIW IIPERNVIGT TPQDFHPPTS LKNGDSSYYD PNYLQSDEEK DRFLKIVTKI FNRINNNLSG GILLEELSKA NPYLGNDNTP DNQFHIGDAS AVEIKFSNGS QDILLPNVII MGAEPDLFET NSSNISLRNN YMPSNHRFGS IAIVTFSPEY SFRFNDNCMN EFIQDPALTL MHELIHSLHG LYGAKGITTK YTITQKQNPL ITNIRGTNIE EFLTFGGTDL NIITSAQSND IYTNLLADYK KIASKLSKVQ VSNPLLNPYK DVFEAKYGLD KDASGIYSVN INKFNDIFKK LYSFTEFDLR TKFQVKCRQT YIGQYKYFKL SNLLNDSIYN ISEGYNINNL KVNFRGQNAN LNPRIITPIT GRGLVKKIIR FCKNIVSVKG IRKSICIEIN NGELFFVASE NSYNDDNINT PKEIDDTVTS NNNYENDLDQ VILNFNSESA PGLSDEKLNL TIQNDAYIPK YDSNGTSDIE QHDVNELNVF FYLDAQKVPE GENNVNLTSS IDTALLEQPK IYTFFSSEFI NNVNKPVQAA LFVSWIQQVL VDFTTEANQK STVDKIADIS IVVPYIGLAL NIGNEAQKGN FKDALELLGA GILLEFEPEL LIPTILVFTI KSFLGSSDNK NKVIKAINNA LKERDEKWKE VYSFIVSNWM TKINTQFNKR KEQMYQALQN QVNAIKTIIE SKYNSYTLEE KNELTNKYDI KQIENELNQK VSIAMNNIDR FLTESSISYL MKIINEVKIN KLREYDENVK TYLLNYIIQH GSILGESQQE LNSMVTDTLN NSIPFKLSSY TDDKILISYF NKFFKRIKSS SVLNMRYKND KYVDTSGYDS NININGDVYK YPTNKNQFGI YNDKLSEVNI SQNDYIIYDN KYKNFSISFW VRIPNYDNKI VNVNNEYTII NCMRDNNSGW KVSLNHNEII WTFEDNRGIN QKLAFNYGNA NGISDYINKW IFVTITNDRL GDSKLYINGN LIDQKSILNL GNIHVSDNIL FKIVNCSYTR YIGIRYFNIF DKELDETEIQ TLYSNEPNTN ILKDFWGNYL LYDKEYYLLN VLKPNNFIDR RKDSTLSINN IRSTILLANR LYSGIKVKIQ RVNNSSTNDN LVRKNDQVYI NFVASKTHLF PLYADTATTN KEKTIKISSS GNRFNQVVVM NSVGNCTMNF KNNNGNNIGL LGFKADTVVA STWYYTHMRD HTNSNGCFWN FISEEHGWQE K

[0318] 6. Botulinum Neurotoxin Type F (BoNT/F) (EC 3.4.24.69) (Bontoxilysin-F)

TABLE-US-00029 P30996; BXF_CLOBO (SEQ ID NO: 144) MPVAINSFNY NDPVNDDTIL YMQIPYEEKS KKYYKAFEIM RNVWIIPERN TIGTNPSDFD PPASLKNGSS AYYDPNYLTT DAEKDRYLKT TIKLFKRINS NPAGKVLLQE ISYAKPYLGN DHTPIDEFSP VTRTTSVNIK LSTNVESSML LNLLVLGAGP DIFESCCYPV RKLIDPDVVY DPSNYGFGSI NIVTFSPEYE YTFNDISGGH NSSTESFIAD PAISLAHELI HALHGLYGAR GVTYEETIEV KQAPLMIAEK PIRLEEFLTF GGQDLNIITS AMKEKIYNNL LANYEKIATR LSEVNSAPPE YDINEYKDYF QWKYGLDKNA DGSYTVNENK FNEIYKKLYS FTESDLANKF KVKCRNTYFI KYEFLKVPNL LDDDIYTVSE GFNIGNLAVN NRGQSIKLNP KIIDSIPDKG LVEKIVKFCK SVIPRKGTKA PPRLCIRVNN SELFFVASES SYNENDINTP KEIDDTTNLN NNYRNNLDEV ILDYNSQTIP QISNRTLNTL VQDNSYVPRY DSNGTSEIEE YDVVDFNVFF YLHAQKVPEG ETNISLTSSI DTALLEESKD IFFSSEFIDT INKPVNAALF IDWISKVIRD FTTEATQKST VDKIADISLI VPYVGLALNI IIEAEKGNFE EAFELLGVGI LLEFVPELTI PVILVFTIKS YIDSYENKNK AIKAINNSLI EREAKWKEIY SWIVSNWLTR INTQFNKRKE QMYQALQNQV DAIKTAIEYK YNNYTSDEKN RLESEYNINN IEEELNKKVS LAMKNIERFM TESSISYLMK LINEAKVGKL KKYDNHVKSD LLNYILDHRS ILGEQTNELS DLVTSTLNSS IPFELSSYTN DKILIIYFNR LYKKIKDSSI LDMRYENNKF IDISGYGSNI SINGNVYIYS TNRNQFGIYN SRLSEVNIAQ NNDIIYNSRY QNFSISFWVR IPKHYKPMNH NREYTIINCM GNNNSGWKIS LRTVRDCEII WTLQDTSGNK ENLIFRYEEL NRISNYINKW IFVTITNNRL GNSRIYINGN LIVEKSISNL GDIHVSDNIL FKIVGCDDET YVGIRYFKVF NTELDKTEIE TLYSNEPDPS ILKNYWGNYL LYNKKYYLFN LLRKDKYITL NSGILNINQQ RGVTEGSVFL NYKLYEGVEV IIRKNGPIDI SNTDNFVRKN DLAYINVVDR GVEYRLYADT KSEKEKIIRT SNLNDSLGQI IVMDSIGNNC TMNFQNNNGS NIGLLGFHSN NLVASSWYYN NIRRNTSSNG CFWSSISKEN GWKE

[0319] 7. Botulinum Neurotoxin Type G (BoNT/G) (EC 3.4.24.69) (Bontoxilysin-G)

TABLE-US-00030 Q60393; BXG_CLOBO (SEQ ID NO: 145) MPVNIKXFNY NDPINNDDII MMEPFNDPGP GTYYKAFRII DRIWIVPERF TYGFQPDQFN ASTGVFSKDV YEYYDPTYLK TDAEKDKFLK TMIKLFNRIN SKPSGQRLLD MIVDAIPYLG NASTPPDKFA ANVANVSINK KIIQPGAEDQ IKGLMTNLII FGPGPVLSDN FTDSMIMNGH SPISEGFGAR MMIRFCPSCL NVFNNVQENK DTSIFSRRAY FADPALTLMH ELIHVLHGLY GIKISNLPIT PNTKEFFMQH SDPVQAEELY TFGGHDPSVI SPSTDMNIYN KALQNFQDIA NRLNIVSSAQ GSGIDISLYK QIYKNKYDFV EDPNGKYSVD KDKFDKLYKA LMFGFTETNL AGEYGIKTRY SYFSEYLPPI KTEKLLDNTI YTQNEGFNIA SKNLKTEFNG QNKAVNKEAY EEISLEHLVI YRIAMCKPVM YKNTGKSEQC IIVNNEDLFF IANKDSFSKD LAKAETIAYN TQNNTIENNF SIDQLILDND LSSGIDLPNE NTEPFTNFDD IDIPVYIKQS ALKKIFVDGD SLFEYLHAQT FPSNIENLQL TNSLNDALRN NNKVYTFFST NLVEKANTVV GASLFVNWVK GVIDDFTSES TQKSTIDKVS DVSIIIPYIG PALNVGNETA KENFKNAFEI GGAAILMEFI PELIVPIVGF FTLESYVGNK GHIIMTISNA LKKRDQKWTD MYGLIVSQWL STVNTQFYTI KERMYNALNN QSQAIEKIIE DQYNRYSEED KMNINIDFND IDFKLNQSIN LAINNIDDFI NQCSISYLMN RMIPLAVKKL KDFDDNLKRD LLEYIDTNEL YLLDEVNILK SKVNRHLKDS IPFDLSLYTK DTILIQVFNN YISNISSNAI LSLSYRGGRL IDSSGYGATM NVGSDVIFND IGNGQFKLNN SENSNITAHQ SKFVVYDSMF DNFSINFWVR TPKYNNNDIQ TYLQNEYTII SCIKNDSGWK VSIKGNRIIW TLIDVNAKSK SIFFEYSIKD NISDYINKWF SITITNDRLG NANIYINGSL KKSEKILNLD RINSSNDIDF KLINCTDTTK FVWIKDFNIF GRELNATEVS SLYWIQSSTN TLKDFWGNPL RYDTQYYLFN QGMQNIYIKY FSKASMGETA PRTNFNNAAI NYQNLYLGLR FIIKKASNSR NINNDNIVRE GDYIYLNIDN ISDESYRVYV LVNSKEIQTQ LFLAPINDDP TFYDVLQIKK YYEKTTYNCQ ILCEKDTKTF GLFGIGKFVK DYGYVWDTYD NYFCISQWYL RRISENINKL RLGCNWQFIP VDEGWTE

[0320] 8. Tetanus Toxin (EC 3.4.24.68) (Tentoxylysin)

TABLE-US-00031 P04958; TETX_CLOTE (SEQ ID NO: 146) MPITINNFRY SDPVNNDTII MMEPPYCKGL DIYYKAFKIT DRIWIVPERY EFGTKPEDFN PPSSLIEGAS EYYDPNYLRT DSDKDRFLQT MVKLFNRIKN NVAGEALLDK IINAIPYLGN SYSLLDKFDT NSNSVSFNLL EQDPSGATTK SAMLTNLIIF GPGPVLNKNE VRGIVLRVDN KNYFPCRDGF GSIMQMAFCP EYVPTFDNVI ENITSLTIGK SKYFQDPALL LMHELIHVLH GLYGMQVSSH EIIPSKQEIY MQHTYPISAE ELFTFGGQDA NLISIDIKND LYEKTLNDYK AIANKLSQVT SCNDPNIDID SYKQIYQQKY QFDKDSNGQY IVNEDKFQIL YNSIMYGFTE IELGKKFNIK TRLSYFSMNH DPVKIPNLLD DTIYNDTEGF NIESKDLKSE YKGQNMRVNT NAFRNVDGSG LVSKLIGLCK KIIPPTNIRE NLYNRTASLT DLGGELCIKI KNEDLTFIAE KNSFSEEPFQ DEIVSYNTKN KPLNFNYSLD KIIVDYNLQS KITLPNDRTT PVTKGIPYAP EYKSNAASTI EIHNIDDNTI YQYLYAQKSP TTLQRITMTN SVDDALINST KIYSYFPSVI SKVNQGAQGI LFLQWVRDII DDFTNESSQK TTIDKISDVS TIVPYIGPAL NIVKQGYEGN FIGALETTGV VLLLEYIPEI TLPVIAALSI AESSTQKEKI IKTIDNFLEK RYEKWIEVYK LVKAKWLGTV NTQFQKRSYQ MYRSLEYQVD AIKKIIDYEY KIYSGPDKEQ IADEINNLKN KLEEKANKAM ININIFMRES SRSFLVNQMI NEAKKQLLEF DTQSKNILMQ YIKANSKFIG ITELKKLESK INKVFSTPIP FSYSKNLDCW VDNEEDIDVI LKKSTILNLD INNDIISDIS GFNSSVITYP DAQLVPGING KAIHLVNNES SEVIVHKAMD IEYNDMFNNF TVSFWLRVPK VSASHLEQYG TNEYSIISSM KKHSLSIGSG WSVSLKGNNL IWTLKDSAGE VRQITFRDLP DKFNAYLANK WVFITITNDR LSSANLYING VLMGSAEITG LGAIREDNNI TLKLDRCNNN NQYVSIDKFR IFCKALNPKE IEKLYTSYLS ITFLRDFWGN PLRYDTEYYL IPVASSSKDV QLKNITDYMY LTNAPSYTNG KLNIYYRRLY NGLKFIIKRY TPNNEIDSFV KSGDFIKLYV SYNNNEHIVG YPKDGNAFNN LDRILRVGYN APGIPLYKKM EAVKLRDLKT YSVQLKLYDD KNASLGLVGT HNGQIGNDPN RDILIASNWY FNHLKDKILG CDWYFVPTDE GWTND

[0321] 9. Diphtheria Toxin (DT) (NAD(+)-diphthamide ADP-ribosyltransferase) (EC 2.4.2.36)

TABLE-US-00032 P00588; DTX_CORBE (SEQ ID NO: 147) MLVRGYVVSR KLFASILIGA LLGIGAPPSA HAGADDVVDS SKSFVMENFS SYHGTKPGYV DSIQKGIQKP KSGTQGNYDD DWKGFYSTDN KYDAAGYSVD NENPLSGKAG GVVKVTYPGL TKVLALKVDN AETIKKELGL SLTEPLMEQV GTEEFIKRFG DGASRVVLSL PFAEGSSSVE YINNWEQAKA LSVELEINFE TRGKRGQDAM YEYMAQACAG NRVRRSVGSS LSCINLDWDV IRDKTKTKIE SLKEHGPIKN KMSESPNKTV SEEKAKQYLE EFHQTALEHP ELSELKTVTG TNPVFAGANY AAWAVNVAQV IDSETADNLE KTTAALSILP GIGSVMGIAD GAVHHNTEEI VAQSIALSSL MVAQAIPLVG ELVDIGFAAY NFVESIINLF QVVHNSYNRP AYSPGHKTQP FLHDGYAVSW NTVEDSIIRT GFQGESGHDI KITAENTPLP IAGVLLPTIP GKLDVNKSKT HISVNGRKIR MRCRAIDGDV TFCRPKSPVY VGNGVHANLH VAFHRSSSEK IHSNEISSDS IGVLGYQKTV DHTKVNSKLS LFFEIKS

[0322] 10. Pseudomonas Exotoxin

TABLE-US-00033 P11439; TOXA_PSEAE (SEQ ID NO: 148) MHLTPHWIPL VASLGLLAGG SFASAAEEAF DLWNECAKAC VLDLKDGVRS SRMSVDPAIA DTNGQGVLHY SMVLEGGNDA LKLAIDNALS ITSDGLTIRL EGGVEPNKPV RYSYTRQARG SWSLNWLVPI GHEKPSNIKV FIHELNAGNQ LSHMSPIYTI EMGDELLAKL ARDATFFVRA HESNEMQPTL AISHAGVSVV MAQAQPRREK RWSEWASGKV LCLLDPLDGV YNYLAQQRCN LDDTWEGKIY RVLAGNPAKH DLDIKPTVIS HRLHFPEGGS LAALTAHQAC HLPLETFTRH RQPRGWEQLE QCGYPVQRLV ALYLAARLSW NQVDQVIRNA LASPGSGGDL GEAIREQPEQ ARLALTLAAA ESERFVRQGT GNDEAGAASA DVVSLTCPVA AGECAGPADS GDALLERNYP TGAEFLGDGG DISFSTRGTQ NWTVERLLQA HRQLEERGYV FVGYHGTFLE AAQSIVFGGV RARSQDLDAI WRGFYIAGDP ALAYGYAQDQ EPDARGRIRN GALLRVYVPR SSLPGFYRTG LTLAAPEAAG EVERLIGHPL PLRLDAITGP EEEGGRLETI LGWPLAERTV VIPSAIPTDP RNVGGDLDPS SIPDKEQAIS ALPDYASQPG KPPREDLK

[0323] Peptide Synthesis

[0324] There are at least four ways to obtain a peptide: (1) purification from a biological system (e.g., tissue, serum, urine, etc.); (Donini P et al., Acta Endocrinol (Copenh). 1966; 52(2):169-85 and Donini P et al., Acta Endocrinol (Copenh). 1966; 52(2):186-98. (2) purification of a peptide fragment after digestion of a protein; (Schulz-Knappe P et al., Eur J Med. Res. 1996; 1(5):223-36. and Kilara A, and Panyam D. Crit. Rev Food Sci Nutr. 2003; 43(6):607-33 (3) genetic engineering and recombinant technologies well known in the art (Martial J A et al., Science. 1979; 205(4406):602-7) and (4) direct chemical synthesis Peptide Synthesis and Applications, 1984. Edited by John Howl (Methods in Molecular Biology, Vol. 298), Humana Press, Totowa, N.J. Chemistry of Peptide Synthesis, 2005. N. Leo Benoiton, CRC Press, Boca Raton, Fla.

[0325] The first two approaches are often impractical due to a lack of control over the peptide sequences. The first approach is also problematic due to a low concentration of peptide in biological samples that requires significant concentrating steps prior to purification. Typically, therefore, for shorter peptides direct chemical synthesis is an attractive option, whereas, for larger peptides, recombinant technology is preferred.

[0326] Traditional synthetic approaches of organic chemistry are generally impractical for peptides with more than four or five amino acid residues due to the complexities of amino acids and peptides. The problems include the presence of multiple reactive groups in the peptide which lead to multiple sites of conjugation on a peptide thereby leading to peptide mixtures that are impure with respect to the peptide of interest and therefore require purification after each step. (Reference: Lehninger Principles of Biochemistry, 3.sup.rd Ed., 2000. Edited by David L. Nelson and Michael M. Cox, Worth Publishers, New York, N.Y.)

[0327] The advent of solid phase peptide synthesis (Merrifield, 1962) in which a peptide is synthesized while keeping it immobilized at one end to a solid support provided the major breakthrough in the direct chemical synthesis of peptides. Today, most solid phase peptide syntheses involve FMOC chemistry. Briefly, chemical synthesis proceeds from the carboxyl terminus (C terminus) to the amino terminus (N terminus). The solid phase support is an insoluble polymer or resin. The 9-fluorenyl-methoxycarbonyl (FMOC) group prevents unwanted reactions at the .alpha.-amino group of the amino acid residue. The peptide is built on a resin support one amino acid at a time using a standard set of reactions in a repeating cycle. First, the C-terminal amino acid with the .alpha.-amino group protected by an FMOC group is attached to the reactive group on the resin. The protecting group on the .alpha.-amino group of the amino acid attached to the resin is removed, generally with a mild organic base. Now, the resin with the C-terminal amino acid is ready to receive the second amino acid of the peptide. Each amino acid is received, protected with different chemistries at the .alpha.-amino group (FMOC) and carboxyl group (generally, Dicyclohexylcarbodiimide, DCC). The carboxyl group of the second amino acid is activated by removing DCC and reacted with the deprotected .alpha.-amino group of the first amino acid on the solid support to form the peptide bond (Peptide Synthesis and Applications, 1984. Edited by John Howl (Methods in Molecular Biology, Vol. 298), Humana Press, Totowa, N.J. Chemistry of Peptide Synthesis, 2005. N. Leo Benoiton, CRC Press, Boca Raton, Fla.) (Reference: Lehninger Principles of Biochemistry, 3.sup.rd Ed., 2000. Edited by David L. Nelson and Michael M. Cox, Worth Publishers, New York, N.Y.).

[0328] At each successive step in the cycle, protective chemical groups block unwanted reactions and the sequence of (i) deprotection of the .alpha.-amino group on the nascent peptide; (ii) activation of the carboxyl group on the next amino acid and (iii) reaction to form peptide bond continues until the entire peptide sequence is synthesized. When the peptide synthesis is complete, the linkage between the resin and the peptide is cleaved off to obtain the final peptide. The state-of-the-art solid phase peptide synthesis technology is automated, and several kinds of commercial instruments are now available and well known in the art. (Peptide Synthesis and Applications, 1984. Edited by John Howl (Methods in Molecular Biology, Vol. 298), Humana Press, Totowa, N.J. Chemistry of Peptide Synthesis, 2005. N. Leo Benoiton, CRC Press, Boca Raton, Fla.; Lehninger Principles of Biochemistry, 3.sup.rd Ed., 2000. Edited by David L. Nelson and Michael M. Cox, Worth Publishers, New York, N.Y.)

[0329] Since the solid phase synthesis is a stepwise process for longer peptides it has the important limitation of lower overall yield and therefore increased cost. For example, with a 96% stepwise yield, the overall yield for 21mer, 51mer and 100mer peptides are 44%, 13% and 1.7%, respectively. Similarly, with a 99.8% stepwise yield, the overall yield for 21mer, 51mer and 100mer peptides are 96%, 90% and 82%, respectively. Therefore, for longer peptides it is more cost- and time-effective to genetically engineer A sequence in an expression cassette and express the sequence in an appropriate expression system (e.g., microbial expression system such as E. coli or yeast) or mammalian expression system (cell culture). For smaller peptides, however, the cost of genetically engineer the sequence and expressing and purifying the peptides are generally cost- and time-effective compared to the solid phase peptide synthesis.

[0330] Peptides useful for the current invention are synthesized, expressed or purified using the methods described above or other methods of synthesis, expression or purification known in the art.

[0331] Formation of dsRNA-Peptide Conjugate

[0332] At least one peptide is conjugated to a dsRNA either to the first or second strand or both and either on the 3' end or 5' end or both or internally. A peptide of the invention can be conjugated to a dsRNA of the invention via any amino acid residue in the peptide, e.g., the C-terminal amino acid of the C-terminus via the carboxyl group of the C-terminal amino acid or the N-terminal amino acid of the N-terminus via the .alpha.-amino group of the N-terminal amino acid or to a specific functional group on the amino acid residue (e.g., --SH group on Cys or amino group of Lys).

[0333] A dsRNA is conjugated to a peptide of the invention using any conjugation chemistry known in the art for peptide or proteins (References: Bioconjugate Techniques, 1996. Greg T. Hermanson, Academic Press, San Diego, Calif.; Chemistry of Protein Conjugation and Cross-linking, 1991. Shan S. Wong, CRC Press, Boca Raton, Fla.).

[0334] In one embodiment the 5' end of the first or second strand is synthesized with a (CH.sub.2).sub.6--NH.sub.3 linker and conjugated to the --SH group of Cys of a peptide using maleimide chemistry to form a stable conjugate.

[0335] In another embodiment the 3' end of the first or second strand is synthesized with a (CH.sub.2).sub.6--SH linker and conjugated to the --SH group of Cys or a peptide via disulfide exchange to form a cleavable conjugate.

[0336] Following conjugation, dsRNA-peptide conjugates are purified by methods well known in the art (Oehlke J et al., Eur J. Biochem. 2002; 269(16):4025-32, Hamma T and Miller P S. Bioconjug Chem. 2003; 14(2):320-30, Zatsepin T S et al., Bioconjug Chem. 2005; 16(3):471-89, Ferenc G et al. Nucleosides Nucleotides Nucleic Acids. 2005; 24(5-7):1059-61). and characterized for identity and purity with standard analytical methods.

[0337] Determining the Function of dsRNA-Delivery Peptide Conjugates

[0338] A dsRNA-peptide conjugate of the invention is assayed to determine the ability of the dsRNA to be delivered to the appropriate target and to mediate RNAi cleavage (as described in the section entitled "RNAi In Vitro Assay to Assess DsiRNA Activity", hereinbelow). A dsRNA peptide conjugate of the invention is also assayed to determine the ability of the peptide to be delivered to the appropriate target.

[0339] In one embodiment, a dsRNA-peptide or a peptide alone attaches to or interacts with a cell surface. The dsRNA-peptide conjugates or the peptide alone is taken up by a cell by directly penetrating the cell membrane, by an endocytic pathway, by both or by other methods known in the art.

[0340] The functionality of a dsRNA-peptide conjugate of the invention can be determined by quantitation of dsRNA Oligonucleotide according to the following method.

[0341] The technology employed to quantitate the DsiRNA oligonucleotides from plasma or tissue samples consists of solid phase extraction to isolate the analyte from the matrix followed by reversed phase ion pairing ultraperformance liquid chromatography (HPLC) separation and detection by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The analytical instrumentation consists of a Waters Acquity HPLC chromatograph with a photodiode array detector connected in series to a Waters Quattro Premiere triple quadrupole mass spectrometer.

[0342] The solid phase extraction is accomplished using Phenomenex's Clarity extraction media and protocol. A "load/lysis" buffer is added to the plasma sample containing the oligo to remove any bound proteins. The oligo is preferentially adsorbed onto the solid phase media. Then a series of buffers are used to wash the oligo to remove contaminants and salts which will inhibit separation and ionization. Finally, the oligo is eluted from the media, concentrated and resuspended in a buffer which is amenable to the downstream analysis.

[0343] The chromatographic separation is accomplished using a mobile phase of hexafluoroisopropanol (HFIP) and triethylamine (TEA) and a C.sub.18 stationary phase. The mass spectrometric detection is accomplished using electrospray ionization followed by a tandem MS (MS/MS) analysis. LC/MS system is developed to determine the characteristic transitions for that particular oligonucleotide molecule. Quantitation of the DsiRNA content in the samples is accomplished by comparing the MS response of the samples to a standard curve of the same DsiRNA in the test sample at varied concentrations (Lin et al. J Pharm Biomed Anal. 2007 Jun. 28: 44(2):330-341). The final data is expressed as a concentration of DsiRNA oligo mass per unit volume of sample (e.g., ng/mL).

Modification of DsiRNAs

[0344] dsRNAs and dsRNA-peptide conjugates are transfected in vitro in cell culture models to establish comparative uptake or delivery of the dsRNAs and dsRNA-peptide conjugates. Appropriate cell culture models are utilized and end point measurements include, but are not limited to, one or more of the following: (i) mRNA quantification using qPCR; (ii) protein quantification using Western blot; (iii) labelled cell internalization of dsRNAs and dsRNA-peptide conjugates. Comparative uptake or delivery of the dsRNAs and dsRNA-peptide conjugates are assessed for the amount of delivered dsRNA, the speed of delivery of dsRNA and the stability of delivered dsRNA, for example, using the above-recited end point measurements.

[0345] In one example, transfection is performed in 24- or 48-well plates for transfecting dsRNAs or dsRNA-peptide conjugates into HeLa cells. Prior to application, dsRNAs and dsRNA-peptide conjugates are diluted into the cell culture media and incubated at room temperature for about 30 min. For dose-response experiments, the final concentration of dsRNAs and dsRNA-peptide conjugates applied are varied within a range of 0 to 50 nM. For the time-course experiments, to determine the speed with which a dsRNA is delivered as defined herein, an optimum concentration of dsRNA-peptide conjugate determine from the dose response experiment is studied for various incubation times, e.g., 30 min to 7 days.

[0346] The functionality of peptide, dsRNA and dsRNA-peptide conjugates are also tested by differentially labelling the peptide and the dsRNA with fluorescent tags and performing fluorescent co localization studies. A peptide is tagged with a green fluorescent dye and the dsRNAs are tagged with red florescent dye. Using this methodology, and a comparison with the localization of free (i.e., unconjugated) dsRNA confirms the ability of a peptide to internalize both the peptide alone and dsRNA-peptide conjugates. The following references describe how to conduct fluorescent localization and cellular trafficking studies--Moschos et al., Bioconjug Chem. 2007; 18(5):1450-1459; Moschos et al., Biochemical Society Transactions 2007; 35(4):807-810; Lord-Fontaine et al., J. Neurotrauma 2008; 25:1309-1322; Winton et al., J. Biol. Chem. 2002; 36(6):32820-32829; Lu, Langer and Chen. Mol. Pharm. 2009 Mar. 30. [Epub ahead of print]; McNaughton et al., Proc Natl Acad Sci USA. 2009 Apr. 14; 106(15):6111-6116.

Modification of dsRNAs

[0347] One major factor that inhibits the effect of double stranded RNAs ("dsRNAs") is the degradation of dsRNAs (e.g., double-stranded RNA, siRNAs and DsiRNAs) by nucleases. A 3'-exonuclease is the primary nuclease activity present in serum and modification of the 3'-ends of antisense DNA oligonucleotides is crucial to prevent degradation (Eder et al., 1991, Antisense Res Dev, 1: 141-151). An RNase-T family nuclease has been identified called ERI-1 which has 3' to 5' exonuclease activity that is involved in regulation and degradation of siRNAs (Kennedy et al., 2004, Nature 427: 645-649; Hong et al., 2005, Biochem J, 390: 675-679). This gene is also known as Thex1 (NM.sub.--02067) in mice or THEX1 (NM.sub.--153332) in humans and is involved in degradation of histone mRNA; it also mediates degradation of 3'-overhangs in siRNAs, but does not degrade duplex RNA (Yang et al., 2006, J Biol Chem, 281: 30447-30454). It is therefore reasonable to expect that 3'-end-stabilization of dsRNAs, including the DsiRNAs of the instant invention, will improve stability.

[0348] XRN1 (NM.sub.--019001) is a 5' to 3' exonuclease that resides in P-bodies and has been implicated in degradation of mRNA targeted by miRNA (Rehwinkel et al., 2005, RNA 11: 1640-1647) and may also be responsible for completing degradation initiated by internal cleavage as directed by a siRNA. XRN2 (NM.sub.--012255) is a distinct 5' to 3' exonuclease that is involved in nuclear RNA processing. Although not currently implicated in degradation or processing of siRNAs and miRNAs, these both are known nucleases that can degrade RNAs and may also be important to consider.

[0349] RNase A is a major endonuclease activity in mammals that degrades RNAs. It is specific for ssRNA and cleaves at the 3'-end of pyrimidine bases. SiRNA degradation products consistent with RNase A cleavage can be detected by mass spectrometry after incubation in serum (Turner et al., 2007, Mol Biosyst 3: 43-50). The 3'-overhangs enhance the susceptibility of siRNAs to RNase degradation. Depletion of RNase A from serum reduces degradation of siRNAs; this degradation does show some sequence preference and is worse for sequences having poly A/U sequence on the ends (Haupenthal et al., 2006 Biochem Pharmacol 71: 702-710). This suggests the possibility that lower stability regions of the duplex may "breathe" and offer transient single-stranded species available for degradation by RNase A. RNase A inhibitors can be added to serum and improve siRNA longevity and potency (Haupenthal et al., 2007, Int J. Cancer 121: 206-210).

[0350] In 21mers, phosphorothioate or boranophosphate modifications directly stabilize the internucleoside phosphate linkage. Boranophosphate modified RNAs are highly nuclease resistant, potent as silencing agents, and are relatively non-toxic. Boranophosphate modified RNAs cannot be manufactured using standard chemical synthesis methods and instead are made by in vitro transcription (IVT) (Hall et al., 2004, Nucleic Acids Res 32: 5991-6000; Hall et al., 2006, Nucleic Acids Res 34: 2773-2781). Phosphorothioate (PS) modifications can be easily placed in the RNA duplex at any desired position and can be made using standard chemical synthesis methods. The PS modification shows dose-dependent toxicity, so most investigators have recommended limited incorporation in siRNAs, favoring the 3'-ends where protection from nucleases is most important (Harborth et al., 2003, Antisense Nucleic Acid Drug Dev 13: 83-105; Chiu and Rana, 2003, Mol Cell 10: 549-561; Braasch et al., 2003, Biochemistry 42: 7967-7975; Amarzguioui et al., 2003, Nucleic Acids Research 31: 589-595). More extensive PS modification can be compatible with potent RNAi activity; however, use of sugar modifications (such as 2'-O-methyl RNA) may be superior (Choung et al., 2006, Biochem Biophys Res Commun 342: 919-927).

[0351] A variety of substitutions can be placed at the 2'-position of the ribose which generally increases duplex stability (T.sub.m) and can greatly improve nuclease resistance. 2'-O-methyl RNA is a naturally occurring modification found in mammalian ribosomal RNAs and transfer RNAs. 2'-O-methyl modification in siRNAs is known, but the precise position of modified bases within the duplex is important to retain potency and complete substitution of 2'-O-methyl RNA for RNA will inactivate the siRNA. For example, a pattern that employs alternating 2'-O-methyl bases can have potency equivalent to unmodified RNA and is quite stable in serum (Choung et al., 2006, Biochem Biophys Res Commun 342: 919-927; Czauderna et al., 2003, Nucleic Acids Research 31: 2705-2716).

[0352] The 2'-fluoro (2'-F) modification is also compatible with dsRNA (e.g., siRNA and DsiRNA) function; it is most commonly placed at pyrimidine sites (due to reagent cost and availability) and can be combined with 2'-O-methyl modification at purine positions; 2'-F purines are available and can also be used. Heavily modified duplexes of this kind can be potent triggers of RNAi in vitro (Allerson et al., 2005, J Med Chem 48: 901-904; Prakash et al., 2005, J Med Chem 48: 4247-4253; Kraynack and Baker, 2006, RNA 12: 163-176) and can improve performance and extend duration of action when used in vivo (Morrissey et al., 2005, Hepatology 41: 1349-1356; Morrissey et al., 2005, Nat Biotechnol 23: 1002-1007). A highly potent, nuclease stable, blunt 19mer duplex containing alternative 2'-F and 2'-O-Me bases is taught by Allerson. In this design, alternating 2'-O-Me residues are positioned in an identical pattern to that employed by Czauderna, however the remaining RNA residues are converted to 2'-F modified bases. A highly potent, nuclease resistant siRNA employed by Morrissey employed a highly potent, nuclease resistant siRNA in vivo. In addition to 2'-O-Me RNA and 2'-F RNA, this duplex includes DNA, RNA, inverted abasic residues, and a 3'-terminal PS internucleoside linkage. While extensive modification has certain benefits, more limited modification of the duplex can also improve in vivo performance and is both simpler and less costly to manufacture. Soutschek et al. (2004, Nature 432: 173-178) employed a duplex in vivo and was mostly RNA with two 2'-O-Me RNA bases and limited 3'-terminal PS internucleoside linkages.

[0353] Locked nucleic acids (LNAs) are a different class of 2'-modification that can be used to stabilize dsRNA (e.g., siRNA and DsiRNA). Patterns of LNA incorporation that retain potency are more restricted than 2'-O-methyl or 2'-F bases, so limited modification is preferred (Braasch et al., 2003, Biochemistry 42: 7967-7975; Grunweller et al., 2003, Nucleic Acids Res 31: 3185-3193; Elmen et al., 2005, Nucleic Acids Res 33: 439-447). Even with limited incorporation, the use of LNA modifications can improve dsRNA performance in vivo and may also alter or improve off target effect profiles (Mook et al., 2007, Mol Cancer Ther 6: 833-843).

[0354] Synthetic nucleic acids introduced into cells or live animals can be recognized as "foreign" and trigger an immune response. Immune stimulation constitutes a major class of off-target effects which can dramatically change experimental results and even lead to cell death. The innate immune system includes a collection of receptor molecules that specifically interact with DNA and RNA that mediate these responses, some of which are located in the cytoplasm and some of which reside in endosomes (Marques and Williams, 2005, Nat Biotechnol 23: 1399-1405; Schlee et al., 2006, Mol Ther 14: 463-470). Delivery of siRNAs by cationic lipids or liposomes exposes the siRNA to both cytoplasmic and endosomal compartments, maximizing the risk for triggering a type 1 interferon (IFN) response both in vitro and in vivo (Morrissey et al., 2005, Nat Biotechnol 23: 1002-1007; Sioud and Sorensen, 2003, Biochem Biophys Res Commun 312: 1220-1225; Sioud, 2005, J Mol Biol 348: 1079-1090; Ma et al., 2005, Biochem Biophys Res Commun 330: 755-759). RNAs transcribed within the cell are less immunogenic (Robbins et al., 2006, Nat Biotechnol 24: 566-571) and synthetic RNAs that are immunogenic when delivered using lipid-based methods can evade immune stimulation when introduced unto cells by mechanical means, even in vivo (Heidel et al., 2004, Nat Biotechnol 22: 1579-1582). However, lipid based delivery methods are convenient, effective, and widely used. Some general strategy to prevent immune responses is needed, especially for in vivo application where all cell types are present and the risk of generating an immune response is highest. Use of chemically modified RNAs may solve most or even all of these problems.

[0355] Although certain sequence motifs are clearly more immunogenic than others, it appears that the receptors of the innate immune system in general distinguish the presence or absence of certain base modifications which are more commonly found in mammalian RNAs than in prokaryotic RNAs. For example, pseudouridine, N6-methyl-A, and 2'-O-methyl modified bases are recognized as "self" and inclusion of these residues in a synthetic RNA can help evade immune detection (Kariko et al., 2005, Immunity 23: 165-175). Extensive 2'-modification of a sequence that is strongly immunostimulatory as unmodified RNA can block an immune response when administered to mice intravenously (Morrissey et al., 2005, Nat Biotechnol 23: 1002-1007). However, extensive modification is not needed to escape immune detection and substitution of as few as two 2'-O-methyl bases in a single strand of a siRNA duplex can be sufficient to block a type 1 IFN response both in vitro and in vivo; modified U and G bases are most effective (Judge et al., 2006, Mol Ther 13: 494-505). As an added benefit, selective incorporation of 2'-O-methyl bases can reduce the magnitude of off-target effects (Jackson et al., 2006, RNA 12: 1197-1205). Use of 2'-O-methyl bases should therefore be considered for all dsRNAs intended for in vivo applications as a means of blocking immune responses and has the added benefit of improving nuclease stability and reducing the likelihood of off-target effects.

[0356] Although cell death can result from immune stimulation, assessing cell viability is not an adequate method to monitor induction of IFN responses. IFN responses can be present without cell death, and cell death can result from target knockdown in the absence of IFN triggering (for example, if the targeted gene is essential for cell viability). Relevant cytokines can be directly measured in culture medium and a variety of commercial kits exist which make performing such assays routine. While a large number of different immune effector molecules can be measured, testing levels of IFN-.alpha., TNF-.alpha., and IL-6 at 4 and 24 hours post transfection is usually sufficient for screening purposes. It is important to include a "transfection reagent only control" as cationic lipids can trigger immune responses in certain cells in the absence of any nucleic acid cargo. Including controls for IFN pathway induction should be considered for cell culture work. It is essential to test for immune stimulation whenever administering nucleic acids in vivo, where the risk of triggering IFN responses is highest.

[0357] Modifications can be included in the DsiRNA agents of the present invention so long as the modification does not prevent the DsiRNA agent from serving as a substrate for Dicer. In one embodiment, one or more modifications are made that enhance Dicer processing of the DsiRNA agent. In a second embodiment, one or more modifications are made that result in more effective RNAi generation. In a third embodiment, one or more modifications are made that support a greater RNAi effect. In a fourth embodiment, one or more modifications are made that result in greater potency per each DsiRNA agent molecule to be delivered to the cell. Modifications can be incorporated in the 3'-terminal region, the 5'-terminal region, in both the 3'-terminal and 5'-terminal region or in some instances in various positions within the sequence. With the restrictions noted above in mind, any number and combination of modifications can be incorporated into the DsiRNA agent. Where multiple modifications are present, they may be the same or different. Modifications to bases, sugar moieties, the phosphate backbone, and their combinations are contemplated. Either 5'-terminus can be phosphorylated.

[0358] Examples of modifications contemplated for the phosphate backbone include phosphonates, including methylphosphonate, phosphorothioate, and phosphotriester modifications such as alkylphosphotriesters, and the like. Examples of modifications contemplated for the sugar moiety include 2'-alkyl pyrimidine, such as 2'-O-methyl, 2'-fluoro, amino, and deoxy modifications and the like (see, e.g., Amarzguioui et al., 2003, Nucleic Acids Research 31: 589-595). Examples of modifications contemplated for the base groups include abasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil and the like. Locked nucleic acids, or LNA's, could also be incorporated. Many other modifications are known and can be used so long as the above criteria are satisfied. Examples of modifications are also disclosed in U.S. Pat. Nos. 5,684,143, 5,858,988 and 6,291,438 and in U.S. published patent application No. 2004/0203145 A1. Other modifications are disclosed in Herdewijn (2000, Antisense Nucleic Acid Drug Dev 10: 297-310), Eckstein (2000, Antisense Nucleic Acid Drug Dev 10: 117-21), Rusckowski et al. (2000, Antisense Nucleic Acid Drug Dev 10: 333-345), Stein et al. (2001, Antisense Nucleic Acid Drug Dev 11: 317-25); Vorobjev et al. (2001, Antisense Nucleic Acid Drug Dev 11: 77-85).

[0359] One or more modifications contemplated can be incorporated into either strand. The placement of the modifications in the DsiRNA agent can greatly affect the characteristics of the DsiRNA agent, including conferring greater potency and stability, reducing toxicity, enhance Dicer processing, and minimizing an immune response. In one embodiment, the antisense strand or the sense strand or both strands have one or more 2'-O-methyl modified nucleotides. In another embodiment, the antisense strand contains 2'-O-methyl modified nucleotides. In another embodiment, the antisense stand contains a 3' overhang that is comprised of 2'-O-methyl modified nucleotides. The antisense strand could also include additional 2'-O-methyl modified nucleotides.

[0360] In certain embodiments of the present invention, the DsiRNA agent has one or more properties which enhance its processing by Dicer. According to these embodiments, the DsiRNA agent has a length sufficient such that it is processed by Dicer to produce an active siRNA and at least one of the following properties: (i) the DsiRNA agent is asymmetric, e.g., has a 3' overhang on the antisense strand and (ii) the DsiRNA agent has a modified 3' end on the sense strand to direct orientation of Dicer binding and processing of the dsRNA to an active siRNA. According to this embodiment, the longest strand in the dsRNA comprises 25-35 nucleotides. In one embodiment, the DsiRNA agent is asymmetric such that the sense strand comprises 25-28 nucleotides and the antisense strand comprises 25-30 nucleotides. Thus, the resulting dsRNA has an overhang on the 3' end of the antisense strand. The overhang is 1-4 nucleotides, for example 2 nucleotides. The sense strand may also have a 5' phosphate.

[0361] In other embodiments, the sense strand of the DsiRNA agent is modified for Dicer processing by suitable modifiers located at the 3' end of the sense strand, i.e., the DsiRNA agent is designed to direct orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotides modifiers could include 3'-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T). In one embodiment, deoxynucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the sense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers. In another embodiment, the invention contemplates substituting two DNA bases in the DsiRNA agent to direct the orientation of Dicer processing of the antisense strand. In a further embodiment of the present invention, two terminal DNA bases are substituted for two ribonucleotides on the 3'-end of the sense strand forming a blunt end of the duplex on the 3' end of the sense strand and the 5' end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3'-end of the antisense strand. This is an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.

[0362] The sense and antisense strands of a DsiRNA agent of the instant invention anneal under biological conditions, such as the conditions found in the cytoplasm of a cell. In addition, a region of one of the sequences, particularly of the antisense strand, of the DsiRNA agent has a sequence length of at least 19 nucleotides, wherein these nucleotides are in the 21-nucleotide region adjacent to the 3' end of the antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene.

[0363] The DsiRNA agent may also have one or more of the following additional properties: (a) the antisense strand has a right shift from the typical 21mer, (b) the strands may not be completely complementary, i.e., the strands may contain simple mismatch pairings and (c) base modifications such as locked nucleic acid(s) may be included in the 5' end of the sense strand. A "typical" 21mer siRNA is designed using conventional techniques. In one technique, a variety of sites are commonly tested in parallel or pools containing several distinct siRNA duplexes specific to the same target with the hope that one of the reagents will be effective (Ji et al., 2003, FEBS Lett 552: 247-252). Other techniques use design rules and algorithms to increase the likelihood of obtaining active RNAi effector molecules (Schwarz et al., 2003, Cell 115: 199-208; Khvorova et al., 2003, Cell 115: 209-216; Ui-Tei et al., 2004, Nucleic Acids Res 32: 936-948; Reynolds et al., 2004, Nat Biotechnol 22: 326-330; Krol et al., 2004, J Biol Chem 279: 42230-42239; Yuan et al., 2004, Nucl Acids Res 32 (Webserver issue):W130-134; Boese et al., 2005, Methods Enzymol 392: 73-96). High throughput selection of siRNA has also been developed (U.S. published patent application No. 2005/0042641 A1). Potential target sites can also be analyzed by secondary structure predictions (Heale et al., 2005, Nucleic Acids Res 33(3): e30). This 21mer is then used to design a right shift to include 3-9 additional nucleotides on the 5' end of the 21mer. The sequence of these additional nucleotides may have any sequence. In one embodiment, the added ribonucleotides are based on the sequence of the target gene. Even in this embodiment, full complementarity between the target sequence and the antisense siRNA is not required.

[0364] The first and second oligonucleotides of a DsiRNA agent of the instant invention are not required to be completely complementary. They only need to be substantially complementary to anneal under biological conditions and to provide a substrate for Dicer that produces a siRNA sufficiently complementary to the target sequence. Locked nucleic acids, or LNA's, are well known to a skilled artisan (Elmen et al., 2005, Nucleic Acids Res 33: 439-447; Kurreck et al., 2002, Nucleic Acids Res 30: 1911-1918; Crinelli et al., 2002, Nucleic Acids Res 30: 2435-2443; Braasch and Corey, 2001, Chem Biol 8: 1-7; Bondensgaard et al., 2000, Chemistry 6: 2687-2695; Wahlestedt et al., 2000, Proc Natl Acad Sci USA 97: 5633-5638). In one embodiment, an LNA is incorporated at the 5' terminus of the sense strand. In another embodiment, an LNA is incorporated at the 5' terminus of the sense strand in duplexes designed to include a 3' overhang on the antisense strand.

[0365] In certain embodiments, the DsiRNA agent of the instant invention has an asymmetric structure, with the sense strand having a 25-base pair length, and the antisense strand having a 27-base pair length with a 2 base 3'-overhang. In other embodiments, this DsiRNA agent having an asymmetric structure further contains 2 deoxynucleotides at the 3' end of the sense strand in place of two of the ribonucleotides.

[0366] Certain DsiRNA agent compositions containing two separate oligonucleotides can be linked by a third structure. The third structure will not block Dicer activity on the DsiRNA agent and will not interfere with the directed destruction of the RNA transcribed from the target gene. In one embodiment, the third structure may be a chemical linking group. Many suitable chemical linking groups are known in the art and can be used. Alternatively, the third structure may be an oligonucleotide that links the two oligonucleotides of the DsiRNA agent in a manner such that a hairpin structure is produced upon annealing of the two oligonucleotides making up the dsRNA composition. The hairpin structure will not block Dicer activity on the DsiRNA agent and will not interfere with the directed destruction of the target RNA.

[0367] In certain embodiments, the DsiRNA agents of the invention have several properties which enhance its processing by Dicer. According to such embodiments, the DsiRNA agent has a length sufficient such that it is processed by Dicer to produce an siRNA and at least one of the following properties: (i) the DsiRNA agent is asymmetric, e.g., has a 3' overhang on the sense strand and (ii) the DsiRNA agent has a modified 3' end on the antisense strand to direct orientation of Dicer binding and processing of the dsRNA to an active siRNA. According to these embodiments, the longest strand in the DsiRNA agent comprises 25-30 nucleotides. In one embodiment, the sense strand comprises 25-30 nucleotides and the antisense strand comprises 25-28 nucleotides. Thus, the resulting dsRNA has an overhang on the 3' end of the sense strand. The overhang is 1-4 nucleotides, such as 2 nucleotides. The antisense strand may also have a 5' phosphate.

[0368] In certain embodiments, the sense strand of a DsiRNA agent is modified for Dicer processing by suitable modifiers located at the 3' end of the sense strand, i.e., the DsiRNA agent is designed to direct orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotide modifiers could include 3'-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T). In one embodiment, deoxynucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the sense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers. In another embodiment, the invention contemplates substituting two DNA bases in the dsRNA to direct the orientation of Dicer processing. In a further invention, two terminal DNA bases are located on the 3' end of the sense strand in place of two ribonucleotides forming a blunt end of the duplex on the 5' end of the antisense strand and the 3' end of the sense strand, and a two-nucleotide RNA overhang is located on the 3'-end of the antisense strand. This is an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.

[0369] In certain other embodiments, the antisense strand of a DsiRNA agent is modified for Dicer processing by suitable modifiers located at the 3' end of the antisense strand, i.e., the DsiRNA agent is designed to direct orientation of Dicer binding and processing. Suitable modifiers include nucleotides such as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotide modifiers could include 3'-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-didehydro-2',3'-dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T). In one embodiment, deoxynucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the antisense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers. In another embodiment, the invention contemplates substituting two DNA bases in the dsRNA to direct the orientation of Dicer processing. In a further invention, two terminal DNA bases are located on the 3' end of the antisense strand in place of two ribonucleotides forming a blunt end of the duplex on the 5' end of the sense strand and the 3' end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3'-end of the sense strand. This is also an asymmetric composition with DNA on the blunt end and RNA bases on the overhanging end.

[0370] The sense and antisense strands anneal under biological conditions, such as the conditions found in the cytoplasm of a cell. In addition, a region of one of the sequences, particularly of the antisense strand, of the dsRNA has a sequence length of at least 19 nucleotides, wherein these nucleotides are adjacent to the 3' end of antisense strand and are sufficiently complementary to a nucleotide sequence of the target RNA.

[0371] Additionally, the DsiRNA agent structure can be optimized to ensure that the oligonucleotide segment generated from Dicer's cleavage will be the portion of the oligonucleotide that is most effective in inhibiting gene expression. For example, in one embodiment of the invention, a 27-bp oligonucleotide of the DsiRNA agent structure is synthesized wherein the anticipated 21 to 22-bp segment that will inhibit gene expression is located on the 3'-end of the antisense strand. The remaining bases located on the 5'-end of the antisense strand will be cleaved by Dicer and will be discarded. This cleaved portion can be homologous (i.e., based on the sequence of the target sequence) or non-homologous and added to extend the nucleic acid strand.

[0372] US 2007/0265220 discloses that 27mer DsiRNAs show improved stability in serum over comparable 21mer siRNA compositions, even absent chemical modification. Modifications of DsiRNA agents, such as inclusion of 2'-O-methyl RNA in the antisense strand, in patterns such as detailed above, when coupled with addition of a 5' Phosphate, can improve stability of DsiRNA agents. Addition of 5'-phosphate to all strands in synthetic RNA duplexes may be an inexpensive and physiological method to confer some limited degree of nuclease stability. The chemical modification patterns of the DsiRNA agents of the instant invention are designed to enhance the efficacy of such agents. Accordingly, such modifications are designed to avoid reducing potency of DsiRNA agents; to avoid interfering with Dicer processing of DsiRNA agents; to improve stability in biological fluids (reduce nuclease sensitivity) of DsiRNA agents; or to block or evade detection by the innate immune system. Such modifications are also designed to avoid being toxic and to avoid increasing the cost or impact the ease of manufacturing the instant DsiRNA agents of the invention.

[0373] RNAi In Vitro Assay to Assess DsiRNA Activity

[0374] An in vitro assay that recapitulates RNAi in a cell-free system can be used to evaluate DsiRNA constructs targeting an RNA sequence(s) of interest. The assay comprises the system described by Tuschl et al., 1999, Genes and Development, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use with DsiRNA agents directed against a target RNA. A Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro. Target RNA is generated via in vitro transcription from an appropriate target RNA expressing plasmid using T7 RNA polymerase or via chemical synthesis. Sense and antisense DsiRNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90.degree. C. followed by 1 hour at 37.degree. C., then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide. The Drosophila lysate is prepared using zero to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10% [vol/vol] lysis buffer containing DsiRNA (10 nM final concentration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug/ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 25.degree. C. for 10 minutes before adding RNA, then incubated at 25.degree. C. for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25.times. Passive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which DsiRNA is omitted from the reaction.

[0375] Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [alpha-.sup.32P] CTP, passed over a G50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5'-.sup.32P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by PHOSPHOR IMAGER.RTM. (autoradiography) quantitation of bands representing intact control RNA or RNA from control reactions without DsiRNA and the cleavage products generated by the assay.

[0376] In one embodiment, this assay is used to determine target sites in the RNA target of interest for DsiRNA mediated RNAi cleavage, wherein a plurality of DsiRNA constructs are screened for RNAi mediated cleavage of the RNA target of interest, for example, by analyzing the assay reaction by electrophoresis of labeled target RNA, or by northern blotting, as well as by other methodology well known in the art.

Structures of dsiRNA-Peptide Agents

[0377] In certain embodiments, the dsRNA agents of the invention can have any of the following structures:

[0378] In one such embodiment, the dsRNA comprises:

TABLE-US-00034 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5'

wherein "X"=RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA and "P"=peptide. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0379] In another such embodiment, the dsRNA comprises:

TABLE-US-00035 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5'

wherein "X"=RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA and "P"=peptide. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0380] In another such embodiment, the dsRNA comprises:

TABLE-US-00036 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5'

wherein "X"=RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA and "P"=peptide. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0381] In another such embodiment, the dsRNA comprises:

TABLE-US-00037 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5'

wherein "X"=RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA and "P"=peptide. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0382] In another such embodiment, the dsRNA comprises:

TABLE-US-00038 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5'

wherein "X"=RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA and "P"=peptide. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0383] In another such embodiment, the dsRNA comprises:

TABLE-US-00039 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5'

wherein "X"=RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA and "P"=peptide. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0384] In another such embodiment, the dsRNA comprises:

TABLE-US-00040 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5'

wherein "X"=RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA and "P"=peptide. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0385] In another such embodiment, the dsRNA comprises:

TABLE-US-00041 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5'

wherein "X"=RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA and "P"=peptide. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0386] In another such embodiment, the dsRNA comprises:

TABLE-US-00042 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5'

wherein "X"=RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA and "P"=peptide. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0387] In another such embodiment, the dsRNA comprises:

TABLE-US-00043 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5'

wherein "X"=RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA and "P"=peptide. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0388] In other embodiments, the DsiRNA comprises:

TABLE-US-00044 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5'

or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3'

3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or

5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or

5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5'

or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5'

or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3'

3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or

5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or

5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5'

or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3 ' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3'

3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or

5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5'

or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3'

3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or

5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or

5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5'

or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXDD-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXX-5' or 5'-XXXXXXXXXXXXXXXXXXXXXXXXX-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5' or 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXXXP-5'

wherein "X"=RNA, "X"=2'-O-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, underlined residues are 2'-O-methyl RNA monomers, "D"=DNA and "P"=peptide. The top strand is the sense strand, and the bottom strand is the antisense strand.

[0389] In another embodiment, the dsRNA comprises strands having equal lengths.

[0390] In one such embodiment, the dsRNA comprises:

TABLE-US-00045 5'-XXXXXXXXXXXXXXXXXXXXXXXXXMMP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXMM-5'

wherein "X"=RNA, "M" is Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) and "P"=a peptide. Any such residues of such agents can optionally be 2'-O-methyl RNA monomers-alternating positioning of 2'-O-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above blunt-blunt dsRNA agents.

[0391] In one such embodiment, the dsRNA comprises:

TABLE-US-00046 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXMM-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXMM-5'

wherein "X"=RNA, "M" is Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) and "P"=a peptide. Any such residues of such agents can optionally be 2'-O-methyl RNA monomers-alternating positioning of 2'-O-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above blunt-blunt dsRNA agents.

[0392] In one such embodiment, the dsRNA comprises:

TABLE-US-00047 5'-XXXXXXXXXXXXXXXXXXXXXXXXXMM-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXMMP-5'

wherein "X"=RNA, "M" is Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) and "P"=a peptide. Any such residues of such agents can optionally be 2'-O-methyl RNA monomers-alternating positioning of 2'-O-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above blunt-blunt dsRNA agents.

[0393] In one such embodiment, the dsRNA comprises:

TABLE-US-00048 5'-XXXXXXXXXXXXXXXXXXXXXXXXXMM-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXMM-5'

wherein "X"=RNA, "M" is Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) and "P"=a peptide. Any such residues of such agents can optionally be 2'-O-methyl RNA monomers-alternating positioning of 2'-O-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above blunt-blunt dsRNA agents.

[0394] In one such embodiment, the dsRNA comprises:

TABLE-US-00049 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXMMP-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXMM-5'

wherein "X"=RNA, "M" is Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) and "P"=a peptide. Any such residues of such agents can optionally be 2'-O-methyl RNA monomers-alternating positioning of 2'-O-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above blunt-blunt dsRNA agents.

[0395] In one such embodiment, the dsRNA comprises:

TABLE-US-00050 5'-XXXXXXXXXXXXXXXXXXXXXXXXXMM-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXMMP-5'

wherein "X"=RNA, "M" is Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) and "P"=a peptide. Any such residues of such agents can optionally be 2'-O-methyl RNA monomers-alternating positioning of 2'-O-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above blunt-blunt dsRNA agents.

[0396] In one such embodiment, the dsRNA comprises:

TABLE-US-00051 5'-PXXXXXXXXXXXXXXXXXXXXXXXXXMMP-3' 3'-PXXXXXXXXXXXXXXXXXXXXXXXXXMMP-5'

wherein "X"=RNA, "M" is Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) and "P"=a peptide. Any such residues of such agents can optionally be 2'-O-methyl RNA monomers-alternating positioning of 2'-O-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above blunt-blunt dsRNA agents.

[0397] The invention also contemplates any of the exemplary structures recited below, wherein at least one peptide of the invention is conjugated to at least one end of at least one of the first or second strand or internally to at least one of the first or second strand of the dsRNA of the invention.

[0398] In another embodiment, the DsiRNA comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the DsiRNA, when numbering from the 3'-terminal residue, are mismatched with corresponding residues of the 5'-terminal region on the second strand when first and second strands are annealed to one another). An exemplary 27mer DsiRNA agent with two terminal mismatched residues is shown:

TABLE-US-00052 5'-XXXXXXXXXXXXXXXXXXXXXXXXX.sup.M.sup.M-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXX.sub.M.sub.M-5'

wherein "X"=RNA, "M"=Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M" residues of otherwise complementary strand when strands are annealed. Any of the residues of such agents can optionally be 2'-O-methyl RNA monomers--alternating positioning of 2'-O-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt/fray" DsiRNA agent. The top strand (first strand) is the sense strand, and the bottom strand (second strand) is the antisense strand.

[0399] In certain additional embodiments, the present invention provides compositions for RNA interference (RNAi) that possess one or more base paired deoxyribonucleotides within a region of a double stranded nucleic acid (dsNA) that is positioned 3' of a projected sense strand Dicer cleavage site and correspondingly 5' of a projected antisense strand Dicer cleavage site. The compositions of the invention comprise a dsNA which is a precursor molecule, i.e., the dsNA of the present invention is processed in vivo to produce an active small interfering nucleic acid (siRNA). The dsNA is processed by Dicer to an active siRNA which is incorporated into RISC.

[0400] In certain embodiments, the DsiRNA agents of the invention can have any of the following exemplary structures:

[0401] In one such embodiment, the DsiRNA comprises:

TABLE-US-00053 5'-XXXXXXXXXXXXXXXXXXXXXXXX.sub.N*D.sub.NDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*D.sub.NXX-5'

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, and "N"=1 to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

[0402] In a related embodiment, the DsiRNA comprises:

TABLE-US-00054 5'-XXXXXXXXXXXXXXXXXXXXXXXX.sub.N*D.sub.NDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*D.sub.NDD-5'

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, and "N"=1 to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

[0403] In another such embodiment, the DsiRNA comprises:

TABLE-US-00055 5'-XXXXXXXXXXXXXXXXXXXXXXXX.sub.N*D.sub.NDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*D.sub.NZZ-5'

wherein "X"=RNA, "X"=2'-O-methyl RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=1 to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

[0404] In another such embodiment, the DsiRNA comprises:

TABLE-US-00056 5'-XXXXXXXXXXXXXXXXXXXXXXXX.sub.N*D.sub.NDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*D.sub.NZZ-5'

wherein "X"=RNA, "X"=2'-O-methyl RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=1 to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

[0405] In another embodiment, the DsiRNA comprises:

TABLE-US-00057 5'-XXXXXXXXXXXXXXXXXXXXXXXX.sub.N*[X1/D1].sub.NDD-3' 3'-YXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*[X2/D2].sub.NZZ-5'

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=1 to 50 or more, but is optionally 1-8, where at least one D1.sub.N is present in the top strand and is base paired with a corresponding D2.sub.N in the bottom strand. Optionally, D1.sub.N and D1.sub.N+1 are base paired with corresponding D2.sub.N and D2.sub.N+1; D1.sub.N, D1.sub.N+1 and D1.sub.N+2 are base paired with corresponding D2.sub.N, D1.sub.N+1 and D1.sub.N+2, etc. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

[0406] In any of the above-depicted structures, the 5' end of either the sense strand or antisense strand optionally comprises a phosphate group.

[0407] In another embodiment, the DNA:DNA-extended DsiRNA comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the DsiRNA, when numbering from the 3'-terminal residue, are mismatched with corresponding residues of the 5'-terminal region on the second strand when first and second strands are annealed to one another). An exemplary DNA:DNA-extended DsiRNA agent with two terminal mismatched residues is shown:

TABLE-US-00058 5'-XXXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*D.sub.N.sup.M.sup.M-3' 3'-XXXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*D.sub.NM.sub.M-5'

wherein "X"=RNA, "M"=Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M" residues of otherwise complementary strand when strands are annealed, "D"=DNA and "N"=1 to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. Any of the residues of such agents can optionally be 2'-O-methyl RNA monomers--alternating positioning of 2'-.beta.-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt/fray" DsiRNA agent. In one embodiment, the top strand (first strand) is the sense strand, and the bottom strand (second strand) is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. Modification and DNA:DNA extension patterns paralleling those shown above for asymmetric/overhang agents can also be incorporated into such "blunt/frayed" agents.

[0408] In one embodiment, a length-extended DsiRNA agent is provided that comprises deoxyribonucleotides positioned at sites modeled to function via specific direction of Dicer cleavage, yet which does not require the presence of a base-paired deoxyribonucleotide in the dsNA structure. An exemplary structure for such a molecule is shown:

TABLE-US-00059 5'-XXXXXXXXXXXXXXXXXXXDDXX-3' 3'-YXXXXXXXXXXXXXXXXXDDXXXX-5'

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, and "D"=DNA. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. The above structure is modeled to force Dicer to cleave a minimum of a 21mer duplex as its primary post-processing form. In embodiments where the bottom strand of the above structure is the antisense strand, the positioning of two deoxyribonucleotide residues at the ultimate and penultimate residues of the 5' end of the antisense strand is likely to reduce off-target effects (as prior studies have shown a 2'-O-methyl modification of at least the penultimate position from the 5' terminus of the antisense strand to reduce off-target effects; see, e.g., US 2007/0223427).

[0409] In one embodiment, the DsiRNA comprises:

TABLE-US-00060 5'-D.sub.NXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*Y-3' 3'-D.sub.NXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*-5'

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, and "N"=1 to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

[0410] In a related embodiment, the DsiRNA comprises:

TABLE-US-00061 5'-D.sub.NXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*DD-3' 3'-D.sub.NXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*XX-5'

wherein "X"=RNA, optionally a 2'-O-methyl RNA monomers "D"=DNA, "N"=1 to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand.

[0411] In another such embodiment, the DsiRNA comprises:

TABLE-US-00062 5'-D.sub.NXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*DD-3' 3'-D.sub.NXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*ZZ-5'

wherein "X"=RNA, optionally a 2'-O-methyl RNA monomers "D"=DNA, "N"=1 to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. "Z"=DNA or RNA. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

[0412] In another such embodiment, the DsiRNA comprises:

TABLE-US-00063 5'-D.sub.NZZXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*DD-3' 3'-D.sub.NXXXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*ZZ-5'

wherein "X"=RNA, "X"=2'-O-methyl RNA, "D"=DNA, "Z"=DNA or RNA, and "N"=1 to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

[0413] In another such embodiment, the DsiRNA comprises:

TABLE-US-00064 5'-D.sub.NZZXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*Y-3' 3'-D.sub.NXXXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*-5'

wherein "X"=RNA, "X"=2'-O-methyl RNA, "D"=DNA, "Z"=DNA or RNA, and "N"=1 to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

[0414] In another embodiment, the DsiRNA comprises:

TABLE-US-00065 5'-[X1/D1].sub.NXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*DD-3' 3'-[X2/D2].sub.NXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*ZZ-5'

wherein "X"=RNA, "D"=DNA, "Z"=DNA or RNA, and "N"=1 to 50 or more, but is optionally 1-8, where at least one D1.sub.N is present in the top strand and is base paired with a corresponding D2.sub.N in the bottom strand. Optionally, D1.sub.N and D 1.sub.N+1 are base paired with corresponding D2.sub.N and D2.sub.N+1; D1.sub.N, D1.sub.N+1 and D1.sub.N+2 are base paired with corresponding D2.sub.N, D1.sub.N+1 and D1.sub.N+2, etc. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

[0415] In a related embodiment, the DsiRNA comprises:

TABLE-US-00066 5'-[X1/D1].sub.NXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*Y-3' 3'-[X2/D2].sub.NXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*-5'

wherein "X"=RNA, "D"=DNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, and "N"=1 to 50 or more, but is optionally 1-8, where at least one D1.sub.N is present in the top strand and is base paired with a corresponding D2.sub.N in the bottom strand. Optionally, D1.sub.N and D1.sub.N+1 are base paired with corresponding D2.sub.N and D2.sub.N+1; D1.sub.N; D1.sub.N+1 and D1.sub.N+2 are base paired with corresponding D2.sub.N, D1.sub.N+1 and D1.sub.N+2, etc. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic.

[0416] In any of the above-depicted structures, the 5' end of either the sense strand or antisense strand optionally comprises a phosphate group.

[0417] In another embodiment, the DNA:DNA-extended DsiRNA comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the DsiRNA, when numbering from the 3'-terminal residue, are mismatched with corresponding residues of the 5'-terminal region on the second strand when first and second strands are annealed to one another). An exemplary DNA:DNA-extended DsiRNA agent with two terminal mismatched residues is shown:

TABLE-US-00067 5'-D.sub.NXXXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*.sup.M.sup.M-3' 3'-D.sub.NXXXXXXXXXXXXXXXXXXXXXXXXXX.sub.N*M.sub.M-5'

wherein "X"=RNA, "M"=Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M" residues of otherwise complementary strand when strands are annealed, "D"=DNA and "N"=1 to 50 or more, but is optionally 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. Any of the residues of such agents can optionally be 2'-O-methyl RNA monomers--alternating positioning of 2'-.beta.-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt/fray" DsiRNA agent. In one embodiment, the top strand (first strand) is the sense strand, and the bottom strand (second strand) is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. Modification and DNA:DNA extension patterns paralleling those shown above for asymmetric/overhang agents can also be incorporated into such "blunt/frayed" agents.

[0418] In another embodiment, a length-extended DsiRNA agent is provided that comprises deoxyribonucleotides positioned at sites modeled to function via specific direction of Dicer cleavage, yet which does not require the presence of a base-paired deoxyribonucleotide in the dsNA structure. An exemplary structure for such a molecule is shown:

TABLE-US-00068 5'-XXDDXXXXXXXXXXXXXXXXXXXX.sub.N*Y-3' 3'-DDXXXXXXXXXXXXXXXXXXXXXX.sub.N*-5'

wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers--in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, and "D"=DNA. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. The above structure is modeled to force Dicer to cleave a minimum of a 21mer duplex as its primary post-processing form. In embodiments where the bottom strand of the above structure is the antisense strand, the positioning of two deoxyribonucleotide residues at the ultimate and penultimate residues of the 5' end of the antisense strand is likely to reduce off-target effects (as prior studies have shown a 2'-O-methyl modification of at least the penultimate position from the 5' terminus of the antisense strand to reduce off-target effects; see, e.g., US 2007/0223427).

[0419] In certain embodiments, the "D" residues of any of the above structures include at least one PS-DNA or PS-RNA. Optionally, the "D" residues of any of the above structures include at least one modified nucleotide that inhibits Dicer cleavage.

[0420] While the above-described "DNA-extended" DsiRNA agents can be categorized as either "left extended" or "right extended", DsiRNA agents comprising both left- and right-extended DNA-containing sequences within a single agent (e.g., both flanks surrounding a core dsRNA structure are dsDNA extensions) can also be generated and used in similar manner to those described herein for "right-extended" and "left-extended" agents.

[0421] In some embodiments, the DsiRNA of the instant invention further comprises a linking moiety or domain that joins the sense and antisense strands of a DNA:DNA-extended DsiRNA agent. Optionally, such a linking moiety domain joins the 3' end of the sense strand and the 5' end of the antisense strand. The linking moiety may be a chemical (non-nucleotide) linker, such as an oligomethylenediol linker, oligoethylene glycol linker, or other art-recognized linker moiety. Alternatively, the linker can be a nucleotide linker, optionally including an extended loop and/or tetraloop.

[0422] In one embodiment, the DsiRNA agent has an asymmetric structure, with the sense strand having a 25-base pair length, and the antisense strand having a 27-base pair length with a 1-4 base 3'-overhang (e.g., a one base 3'-overhang, a two base 3'-overhang, a three base 3'-overhang or a four base 3'-overhang). In another embodiment, this DsiRNA agent has an asymmetric structure further containing 2 deoxynucleotides at the 3' end of the sense strand.

[0423] In another embodiment, the DsiRNA agent has an asymmetric structure, with the antisense strand having a 25-base pair length, and the sense strand having a 27-base pair length with a 1-4 base 3'-overhang (e.g., a one base 3'-overhang, a two base 3'-overhang, a three base 3'-overhang or a four base 3'-overhang). In another embodiment, this DsiRNA agent has an asymmetric structure further containing 2 deoxynucleotides at the 3' end of the antisense strand.

[0424] For the above blunt/fray agents of the invention, it is recognized that the precise sequence of a frayed end structure is not critical to efficacy, e.g., one or two of the 3'-terminal residues of the first strand only need to be non-complementary to the corresponding 5'-terminal residues of the second strand. In certain embodiments, the DsiRNA agents of the invention require, e.g., at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or at least 26 residues of the first strand to be complementary to corresponding residues of the second strand. In certain related embodiments, these first strand residues complementary to corresponding residues of the second strand are optionally consecutive residues. Additionally and/or alternatively, certain mismatch residues can also be positioned within, e.g., the 5' half of the sense strand, meaning that in certain embodiments, perfect complementarity between first and second strands does not hold across the entirety of the first and second strands even exclusive of a fray structure at the 3' terminus of the first strand/5' terminus of the second strand--in such embodiments, the first strand can be an effective first strand while the extent of complementarity between first and second strands across residues--optionally exclusive of the two 3'-terminal residues of the first strand/two 5'-terminal residues of the second strand for frayed agents--is at least 80% of residues, at least 85% of residues, at least 90% of residues, at least 90% of residues, at least 96% of residues. In certain embodiments, the extent of complementarity of the second strand of a DsiRNA of the invention relative to the first strand or relative to the target RNA sequence can be a level of complementarity with the first strand or with the target RNA sequence equivalent to that described above for the first strand of such DsiRNAs.

RNA Processing

[0425] siRNA

[0426] The process of siRNA-mediated RNAi is triggered by the presence of long, dsRNA molecules in a cell. During the initiation step of RNAi, these dsRNA molecules are cleaved into 21-23 nucleotide (nt) small-interfering RNA duplexes (siRNAs) by Dicer, a conserved family of enzymes containing two RNase III-like domains (Bernstein et al. 2001; Elbashir et al. 2001). The siRNAs are characterized by a 19-21 base pair duplex region and 2 nucleotide 3' overhangs on each strand. During the effector step of RNAi, the siRNAs become incorporated into a multimeric protein complex called RNA-induced silencing complex (RISC), where they serve as guides to select fully complementary mRNA substrates for degradation. Degradation is initiated by endonucleolytic cleavage of the mRNA within the region complementary to the siRNA. More precisely, the mRNA is cleaved at a position 10 nucleotides from the 5' end of the guiding siRNA (Elbashir et al. 2001 Genes & Dev. 15: 188-200; Nykanen et al. 2001 Cell 107: 309-321; Martinez et al. 2002 Cell 110: 563-574). An endonuclease responsible for this cleavage was identified as Argonaute2 (Ago2; Liu et al. Science, 305: 1437-41).

miRNA

[0427] The majority of human miRNAs (70%)--and presumably the majority of miRNAs of other mammals--are transcribed from introns and/or exons, and approximately 30% are located in intergenic regions (Rodriguez et al., Genome Res. 2004, 14(10A), 1902-1910). In human and animal, miRNAs are usually transcribed by RNA polymerase II (Farh et al. Science 2005, 310(5755), 1817-1821), and in some cases by pol III (Borchert et al. Nat. Struct. Mol. Biol. 2006, 13(12), 1097-1101). Certain viral encoded miRNAs are transcribed by RNA polymerase III (Pfeffer et al. Nat. Methods 2005, 2(4), 269-276; Andersson et al. J. Virol. 2005, 79(15), 9556-9565), and some are located in the open reading frame of viral gene (Pfeffer et al. Nat. Methods 2005, 2(4), 269-276; Samols et al. J. Virol. 2005, 79(14), 9301-9305). miRNA transcription results in the production of large monocistronic, bicistronic or polycistronic primary transcripts (pri-miRNAs). A single pri-miRNA may range from approximately 200 nucleotides (nt) to several kilobases (kb) in length and have both a 5' 7-methylguanosine (m7) caps and a 3' poly (A) tail. Characteristically, the mature miRNA sequences are localized to regions of imperfect stem-loop sequences within the pri-miRNAs (Cullen, Mol. Cell. 2004, 16(6), 861-865).

[0428] The first step of miRNA maturation in the nucleus is the recognition and cleavage of the pri-miRNAs by the RNase III Drosha-DGCR8 nuclear microprocessor complex, which releases a .about.70 nt hairpin-containing precursor molecule called pre-miRNAs, with a monophosphate at the 5' terminus and a 2-nt overhang with a hydroxyl group at the 3' terminus (Cai et al. RNA 2004, 10(12), 1957-1966; Lee et al. Nature 2003, 425(6956), 415-419; Kim Nat. Rev. Mol. Cell. Biol. 2005, 6(5), 376-385). The next step is the nuclear transport of the pre-miRNAs out of the nucleus into the cytoplasm by Exportin-5, a carrier protein (Yi et al. Genes. Dev. 2003, 17(24), 3011-3016, Bohnsack et al. RNA 2004, 10(2), 185-191). Exportin-5 and the GTP-bound form of its cofactor Ran together recognize and bind the 2 nucleotide 3' overhang and the adjacent stem that are characteristics of pre-miRNA (Basyuk et al. Nucl. Acids Res. 2003, 31(22), 6593-6597, Zamore Mol. Cell. 2001, 8(6), 1158-1160). In the cytoplasm, GTP hydrolysis results in release of the pre-miRNA, which is then processed by a cellular endonuclease III enzyme Dicer (Bohnsack et al.). Dicer was first recognized for its role in generating siRNAs that mediate RNA interference (RNAi). Dicer acts in concert with its cofactors TRBP (Transactivating region binding protein; Chendrimata et al. Nature 2005, 436(7051), 740-744) and PACT (interferon-inducible double strand-RNA-dependant protein kinase activator; Lee et al. EMBO J. 2006, 25(3), 522-532). These enzymes bind at the 3' 2 nucleotide overhang at the base of the pre-miRNA hairpin and remove the terminal loop, yielding an approximately 21-nt miRNA duplex intermediate with both termini having 5' monophosphates, 3' 2 nucleotide overhangs and 3' hydroxyl groups. The miRNA guide strand, the 5' terminus of which is energetically less stable, is then selected for incorporation into the RISC(RNA-induced silencing complex), while the `passenger` strand is released and degraded (Maniataki et al. Genes. Dev. 2005, 19(24), 2979-2990; Hammond et al. Nature 2000, 404(6775), 293-296). The composition of RISC remains incompletely defined, but a key component is a member of the Argonaute (Ago) protein family (Maniataki et al.; Meister et al. Mol. Cell. 2004, 15(2), 185-197).

[0429] The mature miRNA then directs RISC to complementary mRNA species. If the target mRNA has perfect complementarity to the miRNA-armed RISC, the mRNA will be cleaved and degraded (Zeng et al. Proc. Natl. Acad. Sci. USA 2003, 100(17), 9779-9784; Hutvagner et al. Science 2002, 297(55 89), 2056-2060). But as the most common situation in mammalian cells, the miRNAs targets mRNAs with imperfect complementarity and suppress their translation, resulting in reduced expression of the corresponding proteins (Yekta et al. Science 2004, 304(5670), 594-596; Olsen et al. Dev. Biol. 1999, 216(2), 671-680). The 5' region of the miRNA, especially the match between miRNA and target sequence at nucleotides 2-7 or 8 of miRNA (starting from position 1 at the 5' terminus), which is called the seed region, is essentially important for miRNA targeting, and this seed match has also become a key principle widely used in computer prediction of the miRNA targeting (Lewis et al. Cell 2005, 120(1), 15-20; Brennecke et al. PLoS Biol. 2005, 3(3), e85). miRNA regulation of the miRNA-mRNA duplexes is mediated mainly through multiple complementary sites in the 3' UTRs, but there are many exceptions. miRNAs may also bind the 5' UTR and/or the coding region of mRNAs, resulting in a similar outcome (Lytle et al. Proc. Natl. Acad. Sci. USA 2007, 104(23), 9667-9672).

RNase H

[0430] RNase H is a ribonuclease that cleaves the 3'-O--P bond of RNA in a DNA/RNA duplex to produce 3'-hydroxyl and 5'-phosphate terminated products. RNase H is a non-specific endonuclease and catalyzes cleavage of RNA via a hydrolytic mechanism, aided by an enzyme-bound divalent metal ion. Members of the RNase H family are found in nearly all organisms, from archaea and prokaryotes to eukaryotes. During DNA replication, RNase H is believed to cut the RNA primers responsible for priming generation of Okazaki fragments; however, the RNase H enzyme may be more generally employed to cleave any DNA:RNA hybrid sequence of sufficient length (e.g., typically DNA:RNA hybrid sequences of 4 or more base pairs in length in mammals).

MicroRNA and MicroRNA-Like Therapeutics

[0431] MicroRNAs (miRNAs) have been described to act by binding to the 3' UTR of a template transcript, thereby inhibiting expression of a protein encoded by the template transcript by a mechanism related to but distinct from classic RNA interference. Specifically, miRNAs are believed to act by reducing translation of the target transcript, rather than by decreasing its stability. Naturally-occurring miRNAs are typically approximately 22 nt in length. It is believed that they are derived from larger precursors known as small temporal RNAs (stRNAs) approximately 70 nt long.

[0432] Interference agents such as siRNAs, and more specifically such as miRNAs, that bind within the 3' UTR (or elsewhere in a target transcript, e.g., in repeated elements of, e.g., Notch and/or transcripts of the Notch family) and inhibit translation may tolerate a larger number of mismatches in the siRNA/template (miRNA/template) duplex, and particularly may tolerate mismatches within the central region of the duplex. In fact, there is evidence that some mismatches may be desirable or required, as naturally occurring stRNAs frequently exhibit such mismatches, as do miRNAs that have been shown to inhibit translation in vitro (Zeng et al., Molecular Cell, 9: 1-20). For example, when hybridized with the target transcript, such miRNAs frequently include two stretches of perfect complementarity separated by a region of mismatch. Such a hybridized complex commonly includes two regions of perfect complementarily (duplex portions) comprising nucleotide pairs, and at least a single mismatched base pair, which may be, e.g., G:A, G:U, G:G, A:A, A:C, U:U, U:C, C:C, G:-, A:-, U:-, C:-, etc. Such mismatched nucleotides, especially if present in tandem (e.g., a two, three or four nucleotide area of mismatch) can form a bulge that separates duplex portions which are located on either flank of such a bulge. A variety of structures are possible. For example, the miRNA may include multiple areas of nonidentity (mismatch). The areas of nonidentity (mismatch) need not be symmetrical in the sense that both the target and the miRNA include nonpaired nucleotides. For example, structures have been described in which only one strand includes nonpaired nucleotides (Zeng et al., FIG. 14). Typically the stretches of perfect complementarily within a miRNA agent are at least 5 nucleotides in length, e.g., 6, 7, or more nucleotides in length, while the regions of mismatch may be, for example, 1, 2, 3, or 4 nucleotides in length.

[0433] In general, any particular siRNA could function to inhibit gene expression both via (i) the "classical" siRNA pathway, in which stability of a target transcript is reduced and in which perfect complementarily between the siRNA and the target is frequently preferred, and also by (ii) the "alternative" pathway (generally characterized as the miRNA pathway in animals), in which translation of a target transcript is inhibited. Generally, the transcripts targeted by a particular siRNA via mechanism (i) would be distinct from the transcript targeted via mechanism (ii), although it is possible that a single transcript could contain regions that could serve as targets for both the classical and alternative pathways. (Note that the terms "classical" and "alternative" are used merely for convenience and generally are believed to reflect historical timing of discovery of such mechanisms in animal cells, but do not reflect the importance, effectiveness, or other features of either mechanism.) One common goal of siRNA design has been to target a single transcript with great specificity, via mechanism (i), while minimizing off-target effects, including those effects potentially elicited via mechanism (ii). However, it is among the goals of the instant invention to provide RNA interference agents that possess mismatch residues by design, either for purpose of mimicking the activities of naturally-occurring miRNAs, or to create agents directed against target RNAs for which no corresponding miRNA is presently known, with the inhibitory and/or therapeutic efficacies/potencies of such "DmiRNA" agents tolerant of, and indeed possibly enhanced by, such mismatches.

[0434] The tolerance of miRNA agents for mismatched nucleotides (and, indeed the existence and natural use of mechanism (ii) above in the cell) suggests the use of miRNAs in manners that are advantageous to and/or expand upon the "classical" use of perfectly complementary siRNAs that act via mechanism (i). Because miRNAs are naturally occurring molecules, there are likely to be distinct advantages in applying miRNAs as therapeutic agents. miRNAs benefit from hundreds of millions of years of evolutionary "fine tuning" of their function. Thus, sequence-specific "off target" effects should not be an issue with naturally occurring miRNAs, nor, by extension, with synthetic DmiRNAs of the invention designed to directly mimic naturally occurring miRNAs. In addition, miRNAs have evolved to modulate the expression of groups of genes, driving both up and down regulation (in certain instances, performing both functions concurrently within a cell with a single miRNA acting promiscuously upon multiple target RNAs), with the result that complex cell functions can be precisely modulated. Such replacement of naturally occurring miRNAs can involve introducing synthetic miRNAs or miRNA mimetics (e.g., DmiRNAs) into diseased tissues in an effort to restore normal proliferation, apoptosis, cell cycle, and other cellular functions that have been affected by down-regulation of one or more miRNAs. In certain instances, reactivation of these miRNA-regulated pathways has produced a significant therapeutic response (e.g., In one study on cardiac hypertrophy, overexpression of miR-133 by adenovirus-mediated delivery of a miRNA expression cassette protected animals from agonist-induced cardiac hypertrophy, whereas reciprocally reduction of miR-133 in wild-type mice by antagomirs caused an increase in hypertrophic markers (Care et al. Nat. Med. 13: 613-618)).

[0435] To date, more than 600 miRNAs have been identified as encoded within the human genome, with such miRNAs expressed and processed by a combination of proteins in the nucleus and cytoplasm. miRNAs are highly conserved among vertebrates and comprise approximately 2% of all mammalian genes. Since each miRNA appears to regulate the expression of multiple, e.g., two, three, four, five, six, seven, eight, nine or even tens to hundreds of different genes, miRNAs can function as "master-switches", efficiently regulating and coordinating multiple cellular pathways and processes. By coordinating the expression of multiple genes, miRNAs play key roles in embryonic development, immunity, inflammation, as well as cellular growth and proliferation.

[0436] Expression and functional studies suggest that the altered expression of specific miRNAs is critical to a variety of human diseases. Mounting evidence indicates that the introduction of specific miRNAs into disease cells and tissues can induce favorable therapeutic responses (Pappas et al., Expert Opin Ther Targets. 12: 115-27). The promise of miRNA therapy is perhaps greatest in cancer due to the apparent role of certain miRNAs as tumor suppressors. The rationale for miRNA-based therapeutics for, e.g., cancer is supported, at least in part, by the following observations: [0437] (1) miRNAs are frequently mis-regulated and expressed at altered levels in diseased tissues when compared to normal tissues. A number of studies have shown altered levels of miRNAs in cancerous tissues relative to their corresponding normal tissues. Often, altered expression is the consequence of genetic mutations that lead to increased or reduced expression of particular miRNAs. Diseases that possess unique miRNA expression signatures can be exploited as diagnostic and prognostic markers, and can be targeted with the DsiRNA (DmiRNA) agents of the invention. [0438] (2) Mis-regulated miRNAs contribute to cancer development by functioning as oncogenes or tumor suppressors. Oncogenes are defined as genes whose over-expression or inappropriate activation leads to oncogenesis. Tumor suppressors are genes that are required to keep cells from being cancerous; the down-regulation or inactivation of tumor suppressors is a common inducer of cancer. Both types of genes represent preferred drug targets, as such targeting can specifically act upon the molecular basis for a particular cancer. Examples of oncogenic miRNAs are miR-155 and miR-17-92; let-7 is an example of a tumor suppressive miRNA. [0439] (3) Administration of miRNA induces a therapeutic response by blocking or reducing tumor growth in pre-clinical animal studies. The scientific literature provides proof-of-concept studies demonstrating that restoring miRNA function can prevent or reduce the growth of cancer cells in vitro and also in animal models. A well-characterized example is the anti-tumor activity of let-7 in models for breast and lung cancer. DsiRNAs (DmiRNAs) of the invention which are designed to mimic let-7 can be used to target such cancers, and it is also possible to use the DsiRNA design parameters described herein to generate new DsiRNA (DmiRNA) agents directed against target RNAs for which no counterpart naturally occurring miRNA is known (e.g., repeats within Notch or other transcripts), to screen for therapeutic lead compounds, e.g., agents that are capable of reducing tumor burden in pre-clinical animal models. [0440] (4) A given miRNA controls multiple cellular pathways and therefore may have superior therapeutic activity. Based on their biology, miRNAs can function as "master switches" of the genome, regulating multiple gene products and coordinating multiple pathways. Genes regulated by miRNAs include genes that encode conventional oncogenes and tumor suppressors, many of which are individually pursued as drug targets by the pharmaceutical industry. Thus, miRNA therapeutics could possess activity superior to siRNAs and other forms of lead compounds by targeting multiple disease and/or cancer-associated genes. Given the observation that mis-regulation of miRNAs is frequently an early event in the process of tumorigenesis, miRNA therapeutics, which replace missing miRNAs, may be the most appropriate therapy. [0441] (5) miRNAs are natural molecules and are therefore less prone to induce non-specific side-effects. Millions of years of evolution helped to develop the regulatory network of miRNAs, fine-tuning the interaction of miRNA with target messenger RNAs. Therefore, miRNAs and miRNA derivatives (e.g., DmiRNAs designed to mimic naturally occurring miRNAs) will have few if any sequence-specific "off-target" effects when applied in the proper context.

[0442] The physical characteristics of siRNAs and miRNAs are similar. Accordingly, technologies that are effective in delivering siRNAs (e.g., DsiRNAs of the invention) are likewise effective in delivering synthetic miRNAs (e.g., DmiRNAs of the invention).

Conjugation and Delivery of DsiRNA Agents

[0443] In certain embodiments the present invention relates to a method for treating a subject having a disease or disorder, or at risk of developing a disease or disorder. In such embodiments, the DsiRNA can act as novel therapeutic agents for controlling the disease or disorder. The method comprises administering a pharmaceutical composition of the invention to the patient (e.g., human), such that the expression, level and/or activity of a target RNA is reduced. The expression, level and/or activity of a polypeptide encoded by an RNA of interest might also be reduced by a DsiRNA of the instant invention, even where said DsiRNA is directed against a non-coding region of the transcript (e.g., a targeted 5' UTR or 3' UTR sequence). Because of their high specificity, the DsiRNAs of the present invention can specifically target a sequence of interest of cells and tissues, optionally in an allele-specific manner where polymorphic alleles exist within an individual and/or population.

[0444] In the treatment of a disease or disorder, the DsiRNA can be brought into contact with the cells or tissue of a subject, e.g., the cells or tissue of a subject exhibiting disregulation of a protein and/or otherwise targeted for reduction of protein levels. For example, DsiRNA substantially identical to all or part of an RNA sequence of interest, may be brought into contact with or introduced into such a cell, either in vivo or in vitro. Similarly, DsiRNA substantially identical to all or part of an RNA sequence of interest may be administered directly to a subject having or at risk of developing a disease or disorder.

[0445] Therapeutic use of the DsiRNA agents of the instant invention can involve use of formulations of DsiRNA agents comprising multiple different DsiRNA agent sequences. For example, two or more, three or more, four or more, five or more, etc. of the presently described agents can be combined to produce a formulation that, e.g., targets multiple different regions of a target RNA, or that not only target an RNA of interest but also target, e.g., cellular target genes associated with a disease or disorder associated with a target RNA of interest. A DsiRNA agent of the instant invention may also be constructed such that either strand of the DsiRNA agent independently targets two or more regions of an RNA target, or such that one of the strands of the DsiRNA agent targets a cellular target gene of a target mRNA known in the art.

[0446] Use of multifunctional DsiRNA molecules that target more then one region of a target nucleic acid molecule can also provide potent inhibition of RNA levels and expression. For example, a single multifunctional DsiRNA construct of the invention can target both.

[0447] Thus, the DsiRNA agents of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat, inhibit, reduce, or prevent a disease or disorder-associated with a target RNA. For example, the DsiRNA molecules can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.

[0448] The DsiRNA molecules also can be used in combination with other known treatments to treat, inhibit, reduce, or prevent a disease or disorder associated with a target RNA in a subject or organism. For example, the described molecules could be used in combination with one or more known compounds, treatments, or procedures to treat, inhibit, reduce, or prevent a disease or disorder associated with a target RNA in a subject or organism as are known in the art.

[0449] A DsiRNA agent of the invention can be conjugated (e.g., at its 5' or 3' terminus of its sense or antisense strand) or unconjugated to another moiety (e.g. a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye, cholesterol, or the like). Modifying DsiRNA agents in this way may improve cellular uptake or enhance cellular targeting activities of the resulting DsiRNA agent derivative as compared to the corresponding unconjugated DsiRNA agent, are useful for tracing the DsiRNA agent derivative in the cell, or improve the stability of the DsiRNA agent derivative compared to the corresponding unconjugated DsiRNA agent.

[0450] The invention also contemplates dsRNA-peptide conjugates further conjugated to a therapeutic agent, for example an agent that treats or ameliorates the symptoms and/or progression of a disease, for example cancer.

Methods of Introducing Nucleic Acids, Vectors, and Host Cells

[0451] DsiRNA agents of the invention may be directly introduced into a cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the nucleic acid. Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the nucleic acid may be introduced.

[0452] The DsiRNA agents of the invention can be introduced using nucleic acid delivery methods known in art including injection of a solution containing the nucleic acid, bombardment by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and the like. The nucleic acid may be introduced along with other components that perform one or more of the following activities: enhance nucleic acid uptake by the cell or otherwise increase inhibition of the target RNA.

[0453] A cell having a target RNA may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like. The cell may be a stem cell or a differentiated cell. Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine or exocrine glands.

[0454] Depending on the particular target RNA sequence and the dose of DsiRNA agent material delivered, this process may provide partial or complete loss of function for the RNA. A reduction or loss of RNA levels or expression (either RNA expression or encoded polypeptide expression) in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of targeted cells is exemplary. Inhibition of RNA levels or expression refers to the absence (or observable decrease) in the level of RNA or RNA-encoded protein. Specificity refers to the ability to inhibit the RNA without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS). Inhibition of target RNA sequence(s) by the DsiRNA agents of the invention also can be measured based upon the effect of administration of such DsiRNA agents upon development/progression of a disease or disorder associated with a target RNA of interest, e.g., tumor formation, growth, metastasis, etc., either in vivo or in vitro. Treatment and/or reductions in tumor or cancer cell levels can include halting or reduction of growth of tumor or cancer cell levels or reductions of, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more, and can also be measured in logarithmic terms, e.g., 10-fold, 100-fold, 1000-fold, 10.sup.5-fold, 10.sup.6-fold, 10.sup.7-fold reduction in cancer cell levels could be achieved via administration of the DsiRNA agents of the invention to cells, a tissue, or a subject.

[0455] For RNA-mediated inhibition in a cell line or whole organism, expression a reporter or drug resistance gene whose protein product is easily assayed can be measured. Such reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin. Depending on the assay, quantitation of the amount of gene expression allows one to determine a degree of inhibition which is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treated according to the present invention.

[0456] Lower doses of injected material and longer times after administration of RNA silencing agent may result in inhibition in a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells). Quantitation of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target RNA or translation of target protein. As an example, the efficiency of inhibition may be determined by assessing the amount of gene product in the cell; RNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory DsiRNA, or translated polypeptide may be detected with an antibody raised against the polypeptide sequence of that region.

[0457] The DsiRNA agent may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of material may yield more effective inhibition; lower doses may also be useful for specific applications.

Pharmaceutical Compositions

[0458] In certain embodiments, the present invention provides for a pharmaceutical composition comprising the dsRNA-peptide agents of the present invention. The dsRNA-peptide agent sample can be suitably formulated and introduced into the environment of the cell by any means that allows for a sufficient portion of the sample to enter the cell to induce gene silencing, if it is to occur. Many formulations for dsRNA are known in the art and can be used so long as the dsRNA gains entry to the target cells so that it can act. See, e.g., U.S. published patent application Nos. 2004/0203145 A1 and 2005/0054598 A1. For example, the dsRNA-peptide agent of the instant invention can be formulated in buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids. Formulations of DsiRNA agent with cationic lipids can be used to facilitate transfection of the DsiRNA agent into cells. For example, cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (published PCT International Application WO 97/30731), can be used. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche) all of which can be used according to the manufacturer's instructions.

[0459] Such compositions typically include the nucleic acid molecule and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0460] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0461] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0462] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0463] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0464] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.

[0465] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0466] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0467] The compounds can also be administered by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (2002), Nature, 418(6893), 38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol., 20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J. Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst. Pharm. 53(3), 325 (1996).

[0468] The compounds can also be administered by any method suitable for administration of nucleic acid agents, such as a DNA vaccine. These methods include gene guns, bio injectors, and skin patches as well as needle-free methods such as the micro-particle DNA vaccine technology disclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermal needle-free vaccination with powder-form vaccine as disclosed in U.S. Pat. No. 6,168,587. Additionally, intranasal delivery is possible, as described in, inter alia, Hamajima et al. (1998), Clin. Immunol. Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in U.S. Pat. No. 6,472,375) and microencapsulation can also be used. Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U.S. Pat. No. 6,471,996).

[0469] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0470] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD.sub.50 (the dose lethal to 50% of the population) and the ED.sub.50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0471] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC.sub.50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0472] As defined herein, a therapeutically effective amount of a nucleic acid molecule (i.e., an effective dosage) depends on the nucleic acid selected. For instance, if a plasmid encoding a DsiRNA agent is selected, single dose amounts in the range of approximately 1 pg to 1000 mg may be administered; in some embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 .mu.g, or 10, 30, 100, or 1000 mg may be administered. In some embodiments, 1-5 g of the compositions can be administered. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[0473] As therapeutically useful peptide according to the invention "increases" targeting, as defined hereinabove, of a peptide-dsRNA conjugate such that less dsRNA (a lower dose of dsRNA) as compared to the amount or dose of an identical dsRNA that is not conjugated to a peptide and that is required to achieve an equivalent level of binding, association or internalization, as determined by the IC.sub.50s in the assays described hereinbelow is required. For example, the IC.sub.50 for a dsRNA-peptide conjugate that is required to achieve a 50% reduction in RNA/gene expression is decreased as compared to the IC.sub.50 for an identical dsRNA that is not conjugated to a peptide, as measured in vivo or in vitro (see for example Hefner et al. J Biomol Tech. 2008 September: 19(4) 231-237; Zimmermann et al. Nature. 2006 May 4: 441(7089):111-114; Durcan et al. Mol. Pharm. 2008 July-August; 5(4):559-566; Heidel et al. Proc Natl Acad Sci USA. 2007 Apr. 3: 104(14):5715-5721.).

[0474] A useful dose of dsRNA-peptide as defined herein is on the order of 0.1 mg/kg-100 mg/kg, for example, 0.2 kg/mg-50 kg/mg, 0.5 kg/mg-30 kg/mg or 0.5 mg/kg-20 mg/kg (including 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20 kg/mg or more).

[0475] The nucleic acid molecules of the invention can be inserted into expression constructs, e.g., viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, e.g., using methods known in the art, including but not limited to those described in Xia et al., (2002), supra. Expression constructs can be delivered to a subject by, for example, inhalation, orally, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91, 3054-3057). The pharmaceutical preparation of the delivery vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the delivery vehicle is imbedded. Alternatively, where the complete delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0476] The expression constructs may be any construct suitable for use in the appropriate expression system and include, but are not limited to retroviral vectors, linear expression cassettes, plasmids and viral or virally-derived vectors, as known in the art. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems such as U6 snRNA promoters or H1 RNA polymerase III promoters, or other promoters known in the art. The constructs can include one or both strands of the siRNA. Expression constructs expressing both strands can also include loop structures linking both strands, or each strand can be separately transcribed from separate promoters within the same construct. Each strand can also be transcribed from a separate expression construct, e.g., Tuschl (2002, Nature Biotechnol 20: 500-505).

[0477] It can be appreciated that the method of introducing DsiRNA agents into the environment of the cell will depend on the type of cell and the make up of its environment. For example, when the cells are found within a liquid, one preferable formulation is with a lipid formulation such as in lipofectamine and the DsiRNA agents can be added directly to the liquid environment of the cells. Lipid formulations can also be administered to animals such as by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as are known in the art. When the formulation is suitable for administration into animals such as mammals and more specifically humans, the formulation is also pharmaceutically acceptable. Pharmaceutically acceptable formulations for administering oligonucleotides are known and can be used. In some instances, it may be preferable to formulate DsiRNA agents in a buffer or saline solution and directly inject the formulated DsiRNA agents into cells, as in studies with oocytes. The direct injection of DsiRNA agents duplexes may also be done. For suitable methods of introducing dsRNA (e.g., DsiRNA agents), see U.S. published patent application No. 2004/0203145 A1.

[0478] Suitable amounts of a DsiRNA agent must be introduced and these amounts can be empirically determined using standard methods. Typically, effective concentrations of individual DsiRNA agent species in the environment of a cell will be about 50 nanomolar or less, 10 nanomolar or less, or compositions in which concentrations of about 1 nanomolar or less can be used. In another embodiment, methods utilizing a concentration of about 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less and even a concentration of about 10 picomolar or less, 5 picomolar or less, 2 picomolar or less or 1 picomolar or less can be used in many circumstances.

[0479] The method can be carried out by addition of the DsiRNA agent compositions to any extracellular matrix in which cells can live provided that the DsiRNA agent composition is formulated so that a sufficient amount of the DsiRNA agent can enter the cell to exert its effect. For example, the method is amenable for use with cells present in a liquid such as a liquid culture or cell growth media, in tissue explants, or in whole organisms, including animals, such as mammals and especially humans.

[0480] The level or activity of an RNA can be determined by any suitable method now known in the art or that is later developed. It can be appreciated that the method used to measure a target RNA and/or the expression of a target RNA can depend upon the nature of the target RNA. For example, where the target RNA sequence encodes a protein, the term "expression" can refer to a protein or the RNA/transcript derived from the gene of interest (either genomic or of exogenous origin). In such instances the expression of the target RNA can be determined by measuring the amount of target RNA/transcript directly or by measuring the amount of the protein product of the RNA of interest. Protein can be measured in protein assays such as by staining or immunoblotting or, if the protein catalyzes a reaction that can be measured, by measuring reaction rates. All such methods are known in the art and can be used. Where target RNA levels are to be measured, any art-recognized methods for detecting RNA levels can be used (e.g., RT-PCR, Northern Blotting, etc.). In targeting RNAs with the DsiRNA agents of the instant invention, it is also anticipated that measurement of the efficacy of a DsiRNA agent in reducing levels of RNA or protein in a subject, tissue, in cells, either in vitro or in vivo, or in cell extracts can also be used to determine the extent of reduction of phenotypes associated with a particular RNA of interest (e.g., disease or disorders, e.g., cancer or tumor formation, growth, metastasis, spread, etc.). Any of the above measurements can be made on cells, cell extracts, tissues, tissue extracts or any other suitable source material.

[0481] The determination of whether the expression of a target RNA has been reduced can be by any suitable method that can reliably detect changes in RNA levels. Typically, the determination is made by introducing into the environment of a cell undigested DsiRNA such that at least a portion of that DsiRNA agent enters the cytoplasm, and then measuring the level of the target RNA. The same measurement is made on identical untreated cells and the results obtained from each measurement are compared.

[0482] The DsiRNA agent can be formulated as a pharmaceutical composition which comprises a pharmacologically effective amount of a DsiRNA agent and pharmaceutically acceptable carrier. A pharmacologically or therapeutically effective amount refers to that amount of a DsiRNA agent effective to produce the intended pharmacological, therapeutic or preventive result. The phrases "pharmacologically effective amount" and "therapeutically effective amount" or simply "effective amount" refer to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 20% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 20% reduction in that parameter.

[0483] Suitably formulated pharmaceutical compositions of this invention can be administered by any means known in the art such as by parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. In some embodiments, the pharmaceutical compositions are administered by intravenous or intraparenteral infusion or injection.

[0484] In general, a suitable dosage unit of dsRNA will be in the range of 0.001 to 0.25 milligrams per kilogram body weight of the recipient per day, or in the range of 0.01 to 20 micrograms per kilogram body weight per day, or in the range of 0.01 to 10 micrograms per kilogram body weight per day, or in the range of 0.10 to 5 micrograms per kilogram body weight per day, or in the range of 0.1 to 2.5 micrograms per kilogram body weight per day. Pharmaceutical composition comprising the dsRNA can be administered once daily. However, the therapeutic agent may also be dosed in dosage units containing two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage unit. The dosage unit can also be compounded for a single dose over several days, e.g., using a conventional sustained release formulation which provides sustained and consistent release of the dsRNA over a several day period. Sustained release formulations are well known in the art. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose. Regardless of the formulation, the pharmaceutical composition must contain dsRNA in a quantity sufficient to inhibit expression of the target gene in the animal or human being treated. The composition can be compounded in such a way that the sum of the multiple units of dsRNA together contain a sufficient dose.

[0485] Data can be obtained from cell culture assays and animal studies to formulate a suitable dosage range for humans. The dosage of compositions of the invention lies within a range of circulating concentrations that include the ED.sub.50 (as determined by known methods) with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound that includes the IC.sub.50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels of dsRNA in plasma may be measured by standard methods, for example, by high performance liquid chromatography.

[0486] The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.

Methods of Treatment

[0487] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disease or disorder caused, in whole or in part, by the an RNA of interest (e.g., misregulation and/or elevation of transcript and/or protein levels), or treatable via selective targeting of an RNA of interest.

[0488] "Treatment", or "treating" as used herein, is defined as the application or administration of a therapeutic agent (e.g., a DsiRNA agent or vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has the disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward disease.

[0489] In one aspect, the invention provides a method for preventing in a subject, a disease or disorder as described above (including, e.g., prevention of the commencement of transforming events within a subject via inhibition of expression of an RNA of interest), by administering to the subject a therapeutic agent (e.g., a DsiRNA agent or vector or transgene encoding same). Subjects at risk for the disease can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the detection of, e.g., cancer in a subject, or the manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression.

[0490] Another aspect of the invention pertains to methods of treating subjects therapeutically, i.e., altering the onset of symptoms of the disease or disorder. These methods can be performed in vitro (e.g., by culturing the cell with the DsiRNA agent) or, alternatively, in vivo (e.g., by administering the DsiRNA agent to a subject).

[0491] With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. "Pharmacogenomics", as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype"). Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the target RNA molecules of the present invention or target RNA modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[0492] Therapeutic agents can be tested in an appropriate animal model. For example, a DsiRNA agent (or expression vector or transgene encoding same) as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with said agent. Alternatively, an agent (e.g., a therapeutic agent) can be used in an animal model to determine the mechanism of action of such an agent.

Models Useful to Evaluate the Down-Regulation of mRNA Levels and Expression

Cell Culture

[0493] The dsRNA-peptide agents of the invention can be tested for cleavage activity in vivo, for example, using the following procedure.

[0494] The dsRNA-peptide reagents of the invention can be tested in cell culture using HeLa or other mammalian cells to determine the extent of target RNA and target protein inhibition. dsRNA-peptide reagents (e.g., see FIG. 1 and above-recited structures) are selected against the target as described herein. Target RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to, for example, cultured HeLa cells or other transformed or non-transformed mammalian cells in culture. Relative amounts of target RNA are measured versus actin or other appropriate control using real-time PCR monitoring of amplification (e.g., ABI 7700 TAQMAN.RTM.). A comparison is made to a mixture of oligonucleotide sequences made to unrelated targets or to a randomized DsiRNA control with the same overall length and chemistry, but randomly substituted at each position, or simply to appropriate vehicle-treated or untreated controls. Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent concentration is chosen, a RNA time-course of inhibition is performed with the lead DsiRNA molecule.

[0495] TAQMAN.RTM. (Real-Time PCR Monitoring of Amplification) and Lightcycler Quantification of mRNA

[0496] Total RNA is prepared from cells following dsRNA delivery, for example, using Ambion Rnaqueous 4-PCR purification kit for large scale extractions, or Ambion Rnaqueous-96 purification kit for 96-well assays. For Taqman analysis, dual-labeled probes are synthesized with, for example, the reporter dyes FAM or VIC covalently linked at the 5'-end and the quencher dye TAMARA conjugated to the 3'-end. One-step RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence detector using 50 uL reactions consisting of 10 uL total RNA, 100 nM forward primer, 100 mM reverse primer, 100 nM probe, 1.times. TaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl2, 100 uM each dATP, dCTP, dGTP and dTTP, 0.2 U RNase Inhibitor (Promega), 0.025 U AmpliTaq Gold (PE-Applied Biosystems) and 0.2 U M-MLV Reverse Transcriptase (Promega). The thermal cycling conditions can consist of 30 minutes at 48.degree. C., 10 minutes at 95.degree. C., followed by 40 cycles of 15 seconds at 95.degree. C. and 1 minute at 60.degree. C. Quantitation of target KRAS mRNA level is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 30, 10 ng/rxn) and normalizing to, for example, 36B4 mRNA in either parallel or same tube TaqMan reactions.

[0497] Western Blotting

[0498] Nuclear extracts can be prepared using a standard micro preparation technique (see for example Andrews and Faller, 1991, Nucleic Acids Research, 19, 2499). Protein extracts from supernatants are prepared, for example using TCA precipitation. An equal volume of 20% TCA is added to the cell supernatant, incubated on ice for 1 hour and pelleted by centrifugation for 5 minutes. Pellets are washed in acetone, dried and resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts) polyacrylamide gel and transferred onto nitro-cellulose membranes. Non-specific binding can be blocked by incubation, for example, with 5% non-fat milk for 1 hour followed by primary antibody for 16 hours at 4.degree. C. Following washes, the secondary antibody is applied, for example (1:10,000 dilution) for 1 hour at room temperature and the signal detected with SuperSignal reagent (Pierce).

[0499] In several cell culture systems, cationic lipids have been shown to enhance the bioavailability of oligonucleotides to cells in culture (Bennet, et al., 1992, Mol. Pharmacology, 41, 1023-1033). In one embodiment, DsiRNA molecules of the invention are complexed with cationic lipids for cell culture experiments. DsiRNA and cationic lipid mixtures are prepared in serum-free DMEM immediately prior to addition to the cells. DMEM plus additives are warmed to room temperature (about 20-25.degree. C.) and cationic lipid is added to the final desired concentration and the solution is vortexed briefly. DsiRNA molecules are added to the final desired concentration and the solution is again vortexed briefly and incubated for 10 minutes at room temperature. In dose response experiments, the RNA/lipid complex is serially diluted into DMEM following the 10 minute incubation.

Animal Models

[0500] Evaluating the efficacy of dsRNA-peptide agents in animal models is an important prerequisite to human clinical trials. Various animal models of cancer and/or proliferative diseases, conditions, or disorders as are known in the art can be adapted for use for pre-clinical evaluation of the efficacy of DsiRNA compositions of the invention in modulating target gene expression toward therapeutic use.

[0501] For example, if the target is KRAS, as in cell culture models, the most Ras sensitive mouse tumor xenografts are those derived from cancer cells that express mutant Ras proteins. Nude mice bearing H-Ras transformed bladder cancer cell xenografts were sensitive to an anti-Ras antisense nucleic acid, resulting in an 80% inhibition of tumor growth after a 31 day treatment period (Wickstrom, 2001, Mol. Biotechnol., 18, 35-35). Zhang et al., 2000, Gene Ther., 7, 2041, describes an anti-KRAS ribozyme adenoviral vector (KRbz-ADV) targeting a KRAS mutant (KRAS codon 12 GGT to GTT; H441 and H1725 cells respectively). Non-small cell lung cancer cells (NSCLC H441 and H1725 cells) that express the mutant KRas protein were used in nude mouse xenografts compared to NSCLC H1650 cells that lack the relevant mutation. Pre-treatment with KRbz-ADV completely abrogated engraftment of both H441 and H1725 cells and compared to 100% engraftment and tumor growth in animals that received untreated tumor cells or a control vector. Additional mouse models of KRAS misregulation/mutation have also been described (e.g., in Kim et al. Cell 121: 823-835, which identified a role of KRAS in promoting lung adenocarcinomas). The above studies provide proof that inhibition of Ras expression (e.g., KRAS expression) by anti-Ras agents causes inhibition of tumor growth in animals.

[0502] As such, these models can be used in evaluating the efficacy of DsiRNA molecules of the invention in inhibiting KRAS levels, expression, tumor/cancer formation, growth, spread, development of other KRAS-associated phenotypes, diseases or disorders, etc. These models and others can similarly be used to evaluate the safety/toxicity and efficacy of DsiRNA molecules of the invention in a pre-clinical setting.

[0503] The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel et al., 1992), Current Protocols in Molecular Biology (John Wiley & Sons, including periodic updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ. of Oregon Press, Eugene, 2000).

[0504] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES

[0505] The present invention is described by reference to the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.

Example 1

Preparation of Double-Stranded RNA Oligonucleotides

[0506] Oligonucleotide Synthesis and Purification

[0507] DsiRNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein. The DsiRNA molecules are chemically synthesized using methods described herein. Generally, DsiRNA constructs are synthesized using solid phase oligonucleotide synthesis methods as described for 19-23mer siRNAs (see for example Usman et al., U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400; 6,111,086).

[0508] Individual RNA strands are synthesized and HPLC purified according to standard methods (Integrated DNA Technologies, Coralville, Iowa). For example, RNA oligonucleotides are synthesized using solid phase phosphoramidite chemistry, deprotected and desalted on NAP-5 columns (Amersham Pharmacia Biotech, Piscataway, N.J.) using standard techniques (Damha and Olgivie, 1993, Methods Mol Biol 20: 81-114; Wincott et al., 1995, Nucleic Acids Res 23: 2677-84). The oligomers are purified using ion-exchange high performance liquid chromatography (IE-HPLC) on an Amersham Source 15Q column (1.0 cm.times.25 cm; Amersham Pharmacia Biotech, Piscataway, N.J.) using a 15 min step-linear gradient. The gradient varies from 90:10 Buffers A:B to 52:48 Buffers A:B, where Buffer A is 100 mM Tris pH 8.5 and Buffer B is 100 mM Tris pH 8.5, 1 M NaCl. Samples are monitored at 260 nm and peaks corresponding to the full-length oligonucleotide species are collected, pooled, desalted on NAP-5 columns, and lyophilized.

[0509] The purity of each oligomer was determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, Calif.). The CE capillaries had a 100 .mu.m inner diameter and contains ssDNA 100R Gel (Beckman-Coulter). Typically, about 0.6 nmole of oligonucleotide was injected into a capillary, run in an electric field of 444 V/cm and detected by UV absorbance at 260 nm. Denaturing Tris-Borate-7 M-urea running buffer was purchased from Beckman-Coulter. Oligoribonucleotides were obtained that are at least 90% pure as assessed by CE for use in experiments described below. Compound identity was verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy on a Voyager DE.TM. Biospectometry Work Station (Applied Biosystems, Foster City, Calif.) following the manufacturer's recommended protocol. Relative molecular masses of all oligomers were obtained, often within 0.2% of expected molecular mass.

[0510] Preparation of Duplexes

[0511] Single-stranded RNA (ssRNA) oligomers were resuspended, e.g., at 100 .mu.M concentration in duplex buffer consisting of 100 mM potassium acetate, 30 mM HEPES, pH 7.5. Complementary sense and antisense strands were mixed in equal molar amounts to yield a final solution of, e.g., 50 .mu.M duplex. Samples were heated to 100.degree. C. for 5' in RNA buffer (IDT) and allowed to cool to room temperature before use. Double-stranded RNA (dsRNA) oligomers were stored at -20.degree. C. Single-stranded RNA oligomers were stored lyophilized or in nuclease-free water at -80.degree. C.

[0512] Nomenclature

[0513] For consistency, the following nomenclature has been employed in the instant specification. Names given to duplexes indicate the length of the oligomers and the presence or absence of overhangs. A "25/27" is an asymmetric duplex having a 25 base sense strand and a 27 base antisense strand with a 2-base 3'-overhang. A "27/25" is an asymmetric duplex having a 27 base sense strand and a 25 base antisense strand.

[0514] Cell Culture and RNA Transfection

[0515] HeLa cells were obtained from ATCC and maintained in Dulbecco's modified Eagle medium (HyClone) supplemented with 10% fetal bovine serum (HyClone) at 37.degree. C. under 5% CO.sub.2. For RNA transfections, HeLa cells were transfected with DsiRNAs as indicated at a final concentration of 1 nM or 0.1 nM using Lipofectamine.TM. RNAiMAX (Invitrogen) and following manufacturer's instructions. Briefly, 2.54, of a 0.2 .mu.M or 0.02 .mu.M stock solution of each DsiRNA were mixed with 46.54, of Opti-MEM I (Invitrogen) and 1 .mu.L of Lipofectamine.TM. RNAiMAX. The resulting 50 .mu.L mix was added into individual wells of 12 well plates and incubated for 20 min at RT to allow DsiRNA:Lipofectamine.TM. RNAiMAX complexes to form. Meanwhile, HeLa cells were trypsinized and resuspended in medium at a final concentration of 367 cells/.mu.L. Finally, 450 .mu.L of the cell suspension were added to each well (final volume 500 .mu.L) and plates were placed into the incubator for 24 hours.

[0516] Assessment of Inhibition

[0517] Target gene knockdown was determined by qRT-PCR, with values normalized to HPRT expression control treatments, including Lipofectamine.TM. RNAiMAX alone (Vehicle control) or untreated.

[0518] RNA Isolation and Analysis

[0519] Cells were washed once with 2 mL of PBS, and total RNA was extracted using RNeasy Mini Kit.TM. (Qiagen) and eluted in a final volume of 30 .mu.L. 1 .mu.g of total RNA was reverse-transcribed using Transcriptor 1.sup.st Strand cDNA Kit.TM. (Roche) and random hexamers following manufacturer's instructions. One-thirtieth (0.66 .mu.L) of the resulting cDNA was mixed with 54, of IQ Multiplex Powermix (Bio-Rad) together with 3.33 .mu.L of H.sub.2O and 1 .mu.L of a 304 mix containing primers and probes specific for human genes HPRT-1 (accession number NM.sub.--000194) and KRAS target sequences.

[0520] Quantitative RT-PCR

[0521] A CFX96 Real-time System with a C1000 Thermal cycler (Bio-Rad) was used for the amplification reactions. PCR conditions were: 95.degree. C. for 3 min; and then cycling at 95.degree. C., 10 sec; 55.degree. C., 1 min for 40 cycles. Each sample was tested in triplicate. Relative HPRT mRNA levels were normalized to target mRNA levels and compared with mRNA levels obtained in control samples treated with the transfection reagent alone, or untreated. Data was analyzed using Bio-Rad CFX Manager version 1.0 software.

Example 2

Preparation and Use of DsiRNA-Peptide Conjugates

[0522] Oligonucleotide-peptide conjugates of the present invention were synthesized with chemistry based on the conjugation of HyNic (6-Hydrazinonicotinamide)-modified peptides to 4FB (4-Formylbenzamide)-modified oligonucleotides. Other peptide synthesis methods and conjugation procedures known in the art are also applicable.

[0523] HyNic moieties were incorporated on a peptide at either N- or C-termini using 6-Boc-HyNic or FMOC-Lys-(.epsilon.-6-BocHyNic)OH, respectively. Cleavage from resin was accomplished using TFA/acetone/water/triisopropylsilane (TIS) (92.5/2.5/2.5/2.5) for 2 hours. The presence of the acetone forms a hydrazone with the deprotected hydrazine moiety in situ blocking any trifluoroacetamide formation from the reaction of TFA with the strongly nucleophilic hydrazine. Crude peptides were analyzed by HPLC and ES-MS. Products were isolated by RP-HPLC using a gradient method. For Peg12 peptides, polyethylene glycol synthons were directly added during solid phase peptide synthesis. In some instances, additional polyethylene glycol spacers were also added to the oligonucleotide termini using polyethylene glycol oligonucleotide synthons.

[0524] Amino-modified oligonucleotides were converted to 5'-4FB-oligonucleotides. Linking of HyNic-peptides to 4FB-modified oligonucleotides was performed at a 2-5 mole excess of HyNic-peptide and generally produced >80% conjugate yield. Hydrazone bond formation was catalyzed and reaction kinetics improved 10-100-fold via inclusion of aniline, generally leading to conjugation yields >95%. Optimal conjugation kinetics (formation of the hydrazone bond) was achieved between pH 4.5-5.0. However, the reaction also can proceed at higher pH, albeit at a slower rate. The optimum pH for each conjugation was determined empirically, also taking into account the solubility of the different peptide sequences. The degree of conjugation can be monitored spectrophotometrically. Formation of the bis-aryl hydrazone bond was utilized both to trace and to quantify progress of the conjugation reaction, using the known molar extinction coefficient (29,000 @ 354 nm). Diafiltration was used to remove excess peptide, yielding the oligonucleotide-peptide conjugates. To produce HyNic-quenched peptides, HyNic-peptides were reacted with 2-Sulfobenzaldehyde to inactivate the HyNic reactive moiety on the peptide.

[0525] Cell-Free Dicing Assay

[0526] DsiRNA or peptide-conjugated DsiRNA (final concentration at 5 .mu.M) was incubated with recombinant human dicer enzyme mixture (Genlantis, #T52002) at 37.degree. C. for 2 hrs, and the reaction was stopped with stop solution. This final solution was mixed with gel loading buffer (Bio-Rad, #161-0767). Dicer-cleaved dsRNAs and intact DsiRNAs were resolved by 18% native polyacrylamide gel electrophoresis. Gel images were obtained using the Bio-Rad VersaDoc.TM. imaging system (model #4000 MP).

[0527] Serum Stability Assay

[0528] DsiRNA or peptide-conjugated DsiRNA (2 .mu.M final concentration) was incubated in 90% (v/v) mouse serum (Sigma #M5905) at 37.degree. C. At different time points (0, 2, 4, 8, 1, 10 & 25 hours), 10 .mu.L sample was mixed with 2 .mu.L H.sub.2O and 3 .mu.L gel loading buffer (Bio-Rad #161-0767) and was immediately flash frozen in an alcohol-dry ice bath. Samples were electrophoresed on an 18% native polyacrylamide gel (Bio-Rad #161-1216). Resolved siRNA bands were quantified using the Bio-Rad VersaDoc.TM. imaging system (Bio-Rad model #4000 MP). The half-life of individual dsRNAs in 90% serum was calculated by plotting the change in dsRNA band intensity over time.

[0529] HPRT1- and KRAS-Targeting DsiRNAs

[0530] Exemplary DsiRNAs directed against HPRT1 and KRAS target genes were synthesized as described herein, with DsiRNAs possessing the oligonucleotide sequences, 2'-O-methyl and end modifications shown in FIG. 2.

[0531] Conjugated Peptides

[0532] Exemplary peptides used or capable of use in conjugation with the DsiRNAs of the instant invention are listed in FIG. 3, which also indicates the net charge associated with each individual peptide.

[0533] The successful synthesis of various peptide-DsiRNA conjugates was confirmed via observation of the increased size (and, therefore, retarded electrophoretic mobility) associated with a successful conjugation. As shown in FIGS. 4-6, both HPRT1- and KRAS-targeting DsiRNAs were successfully conjugated with a number of peptides, forming the following conjugates: K1379-SEQ ID NO:32; K1379-SEQ ID NO:62; K1459-SEQ ID NO:32 (FIG. 4); K1459-SEQ ID NO:118; K1459-Peg12-SEQ ID NO:118; K1379-SEQ ID NO:118; K1379-Peg12-SEQ ID NO:118 (FIG. 5); H1460-SEQ ID NO:118; and H1460-Peg12-SEQ ID NO:118 (FIG. 6).

[0534] DsiRNA-peptide conjugates possessing cleavable linker moieties (e.g., disulfide groups) were also successfully synthesized. Specifically, FIG. 7 shows that both stable and cleavable conjugates of SEQ ID NOs: 118 and 120 peptides with KRAS-targeting DsiRNA K1379 were produced.

[0535] DsiRNAs were also conjugated with cyclic peptides. As shown in FIG. 8, the KRAS-targeting DsiRNA, K1459, was successfully conjugated with cyclic peptide SEQ ID NO:151.

[0536] To assess Dicer cleavage of certain peptide-DsiRNA conjugates, cell-free dicing assays were performed as described above upon conjugates K1096-SEQ ID NO:39 and K1096-SEQ ID NO:120. As shown in FIGS. 9 and 10, Dicer cleavage of both conjugates was observed.

Example 3

Transfected dsRNA-Peptide Conjugates (e.g., dsRNA-Delivery Peptide Conjugates) Reduced Expression of Target Gene Levels in a Cell

[0537] Cell Culture and RNA Transfection

[0538] HeLa and HepG2 cells were obtained from ATCC and maintained in Dulbecco's modified Eagle medium (HyClone) supplemented with 10% fetal bovine serum (HyClone) at 37.degree. C. under 5% CO.sub.2. For dsRNA and dsRNA-delivery peptide conjugate transfections, HeLa cells were transfected with the unconjugated or conjugated DsiRNAs at indicated final concentrations (e.g., 1 nM or 0.1 nM) in the presence of Lipofectamine.TM. RNAiMAX (Invitrogen). In certain examples, unconjugated DsiRNAs were also used as positive controls. In certain examples, 2.54, of a 0.2 .mu.M or 0.02 .mu.M stock solution of each DsiRNA was mixed with 47.54, of Opti-MEM I (Invitrogen). For Lipofectamine.TM. controls, 2.54, of a 0.2 .mu.M or 0.02 .mu.M stock solution of each DsiRNA was mixed with 46.54, of Opti-MEM I (Invitrogen) and 1 .mu.L of Lipofectamine.TM. RNAiMAX. The resulting 50 .mu.L mix was added into individual wells of 12 well plates and incubated for 20 minutes at room temperature to allow DsiRNA:Lipofectamine.TM. RNAiMAX complexes to form. Meanwhile, HeLa or HepG2 cells were trypsinized and resuspended in medium at a final concentration of about 367 cells/.mu.L. Finally, 450 .mu.L of the cell suspension was added to each well (final volume 500 .mu.L) and plates were placed into the incubator for 24 hours. For dose-response studies, the concentrations of transfected DsiRNAs were varied from initially 1 pM to 1 nM. For dose-response studies involving DsiRNA-peptide conjugates administered to cells in the absence of transfection vehicle, the concentrations of administered DsiRNAs and DsiRNA-peptide conjugates were varied from approximately 5 nM to approximately 5 .mu.M. Time course studies can also be performed, with incubation times of about 4 hours to about 72 hours studied.

[0539] Assessment of Inhibition

[0540] Target gene knockdown was determined by qRT-PCR, with values normalized to HPRT expression control treatments, including Lipofectamine.TM. RNAiMAX alone (Vehicle control) or untreated.

[0541] RNA Isolation and Analysis

[0542] Cells were washed once with 2 mL of PBS, and total RNA was extracted using RNeasy Mini Kit.TM. (Qiagen) and eluted in a final volume of 30 .mu.L. 1 .mu.g of total RNA was reverse-transcribed using Transcriptor 1.sup.st Strand cDNA Kit.TM. (Roche) and random hexamers following manufacturer's instructions. One-thirtieth (0.66 .mu.L) of the resulting cDNA was mixed with 54, of IQ Multiplex Powermix (Bio-Rad) together with 3.334, of H.sub.2O and 14, of a 304 mix containing primers and probes specific for human genes HPRT-1 (accession number NM.sub.--000194) and KRAS target sequences.

[0543] Quantitative RT-PCR

[0544] A CFX96 Real-time System with a C1000 Thermal cycler (Bio-Rad) was used for the amplification reactions. PCR conditions were: 95.degree. C. for 3 min; and then cycling at 95.degree. C., 10 sec; 55.degree. C., 1 min for 40 cycles. Each sample was tested in duplicate (with duplicate experiments performed for each agent for which data is shown in FIGS. 2-19). Relative HPRT mRNA levels were normalized to target mRNA levels and compared with mRNA levels obtained in control samples treated with the transfection reagent alone, or untreated. Data were analyzed using Bio-Rad CFX Manager version 1.0 software. Expression data were presented as a comparison of the expression under the treatment of unconjugated dsRNA versus that of dsRNA-delivery peptide conjugates.

[0545] DsiRNA-peptide conjugates were initially examined for the ability to inhibit target mRNA levels in a cell when administered via transfection. As shown in FIGS. 11 and 12, three different peptide conjugates of KRAS-targeting DsiRNA K1096 conjugates (K1096-SEQ ID NO:39; K1096-Peg12-SEQ ID NO:32; and K1096-SEQ ID NO:120) were all observed to inhibit target mRNA levels by at least 80% when administered to HeLa cells via transfection at 0.1 nM and higher concentrations. The levels of target gene inhibition seen for peptide-DsiRNA conjugates were also observed to be similar to those seen for the free K1096 DsiRNA. Accordingly, peptide-DsiRNA conjugates were observed to be effective inhibitors of target RNA levels when administered to HeLa cells via transfection.

Example 4

DsiRNA-Peptide Conjugates Demonstrated Stability in Serum

[0546] Serum stability of DsiRNA agents was assessed as described herein. As shown in FIGS. 13 and 14, conjugation of DsiRNA K1096 with either the SEQ ID NO:39 or 120 peptide resulted in significantly extended half-lives for conjugated agents K1096-SEQ ID NO:39 (half life=12.1 hours) and K1096-SEQ ID NO:120 (half life=13.3 hours), as compared to DsiRNAs possessing otherwise identical modification patterns but lacking the SEQ ID NO:39 or 120 peptides.

Example 5

dsRNA-Peptide Conjugates (e.g., dsRNA-Delivery Peptide Conjugates) Administered without Transfection Vehicle Reduced Expression of Target Gene Levels in a Cell

[0547] To assess the ability of exemplified DsiRNA-peptide conjugates to promote delivery of such conjugated agents to target cells in the absence of any transfection vehicle, a series of DsiRNA-peptide conjugates were administered to HeLa cells at concentrations ranging from 20 nM to 2 .mu.M. As shown in FIGS. 15-17, elevated concentrations of DsiRNA-peptide conjugates were required to achieve significant inhibition of target mRNA levels, as compared to transfected DsiRNAs. However, all six tested conjugates (K1096-Peg12-SEQ ID NO:32 and K1096-Peg12-SEQ ID NO:62 of FIG. 15; K1096-Peg12-SEQ ID NO:118 and K1096-SEQ ID NO:120 of FIG. 16; and K1336-SEQ ID NO:32 and K1336-SEQ ID NO:62 of FIG. 17) behaved in a dose-responsive manner, with significant levels of target gene inhibition observed for all conjugated molecules at 2 .mu.M concentration. Indeed, the K1096-SEQ ID NO:120 conjugate of FIG. 16 exhibited surprising and significant reduction of target mRNA levels at all tested concentrations (20 nM, 200 nM and 2 .mu.M).

[0548] DsiRNA-peptide conjugates K1379-SEQ ID NO:118 and K1379-SEQ ID NO:120 were then assessed for the ability to inhibit the KRAS target gene when administered to HepG2 cells at a concentration of 5 .mu.M in the absence of transfection vehicle. As shown in FIG. 18, remarkably, greater than 90% reduction in target mRNA levels were observed for both conjugates administered to HepG2 cells at a concentration of 5 .mu.M in the absence of transfection vehicle. Such results were similar to results observed for the K1379 DsiRNA alone administered at 1 nM to HepG2 cells. Surprisingly, inclusion of a quenched peptide (either quenched SEQ ID NO:118 or quenched SEQ ID NO:120) with free DsiRNA K1379 in a mixture that was administered to HepG2 cells resulted in a complete loss of any target mRNA inhibition activity.

[0549] To examine whether exemplary DsiRNA-peptide conjugates were more efficient inhibitors of target mRNA levels in the absence of delivery vehicle than corresponding free DsiRNAs, dose-response curves were obtained that compared free K1379 DsiRNA with K1379-SEQ ID NO:118 conjugate and also compared free K1379 DsiRNA with the K1379-SEQ ID NO:120 conjugate. As shown in FIG. 19, K1379-peptide conjugates performed significantly better than corresponding free K1379 DsiRNAs across all IC.sub.50-informative concentrations. Indeed, measured IC.sub.50 values for DsiRNA-peptide conjugates in the absence of transfection vehicle were two- to three-fold higher (and therefore less potent) for free DsiRNA as compared to corresponding DsiRNA-peptide conjugates.

Example 6

Preparation of Additional Delivery Peptide-dsRNA and/or Targeting Peptide-dsRNA Conjugates

[0550] Additional preferred target DsiRNA agents are selected from a pre-screened population of DsiRNAs. Design of DsiRNAs can optionally involve use of predictive scoring algorithms that perform in silico assessments of the projected activity/efficacy of a number of possible DsiRNAs spanning a region of sequence.

[0551] A dsRNA of the invention is conjugated to a delivery peptide or a targeting peptide by any of the methods described herein above. About 20 mg of DsiRNAs (.about.1 .mu.moles) with 5' amino group are reacted with 3-5 molar excess of peptides with terminal Cys sulfhydryl group using maleimide chemistry (Moschos et al., Bioconjug Chem. 2007; 18(5):1450-9; Nishina et al., Mol. Ther. 2008; 16(4):734-40). The peptide-RNA conjugates are purified by diafiltration to remove excess peptide, desalted and supplied as lyophilized powder. The purity of the final products is determined by analytical anion-exchange HPLC and electrospray mass spectroscopy with deconvolution.

Example 7

Additional Use of a dsRNA-Targeting Peptide Conjugate to Reduce Expression of a Target Gene in a Cell

[0552] Cell Culture and RNA Transfection

[0553] HeLa, Hep3B, HepG2, HT29, LS174T, and Neuro2a are obtained from ATCC and maintained in the recommended basal medium with 10% heat-inactivated FBS at 37.degree. C. under 5% CO.sub.2. For dsRNA and dsRNA-targeting peptide conjugate transfections, cells are transfected with the unconjugated or conjugated DsiRNAs as indicated at a final concentration of 1 nM or 0.1 nM. Lipofectamine.TM. RNAiMAX (Invitrogen). DsiRNAs are used as positive controls. Briefly, 2.5 .mu.L, of a 0.2 .mu.M or 0.02 .mu.M stock solution of each DsiRNAs is mixed with 47.54, of Opti-MEM I (Invitrogen). For Lipofectamine.TM. control, 2.5 .mu.L, of a 0.2 .mu.M or 0.02 .mu.M stock solution of each DsiRNAs is mixed with 46.54, of Opti-MEM I (Invitrogen) and 1 .mu.L of Lipofectamine.TM. RNAiMAX. The resulting 50 .mu.L mix is added into individual wells of 12 well plates and incubated for 20 min at RT to allow DsiRNA:Lipofectamine.TM. RNAiMAX complexes to form. Meanwhile, cells are trypsinized and resuspended in medium at a final concentration of about 367 cells/.mu.L. Finally, 450 .mu.L of the cell suspension are added to each well (final volume 500 .mu.L) and plates are placed into the incubator for 24 hours. For dose response study, the concentrations of DsiRNAs are varied from initially 1 pM to 1 nM. For time course studies, incubation times of about 4 hours to about 72 hours are studied.

[0554] Assessment of Inhibition

[0555] Target gene knockdown is determined by qRT-PCR, with values normalized to HPRT expression control treatments, including Lipofectamine.TM. RNAiMAX alone (Vehicle control) or untreated.

[0556] RNA Isolation and Analysis

[0557] Cells are washed once with 2 mL of PBS, and total RNA is extracted using RNeasy Mini Kit.TM. (Qiagen) and eluted in a final volume of 30 .mu.L. 1 .mu.g of total RNA is reverse-transcribed using Transcriptor 1.sup.st Strand cDNA Kit.TM. (Roche) and random hexamers following manufacturer's instructions. One-thirtieth (0.66 .mu.L) of the resulting cDNA is mixed with 54, of IQ Multiplex Powermix (Bio-Rad) together with 3.334, of H.sub.2) and 14, of a 304 mix containing primers and probes specific for human genes HPRT-1 (accession number NM.sub.--000194) and KRAS target sequences.

[0558] Quantitative RT-PCR

[0559] A CFX96 Real-time System with a C1000 Thermal cycler (Bio-Rad) is used for the amplification reactions. PCR conditions are: 95.degree. C. for 3 min; and then cycling at 95.degree. C., 10 sec; 55.degree. C., 1 min for 40 cycles. Each sample is tested in triplicate. Relative HPRT mRNA levels are normalized to target mRNA levels and compared with mRNA levels obtained in control samples treated with the transfection reagent alone, or untreated. Data are analyzed using Bio-Rad CFX Manager version 1.0 software. Expression data are presented as a comparison of the expression under the treatment of unconjugated dsRNA versus that of dsRNA-targeting peptide conjugates.

Example 8

Use of a dsRNA-Delivery Peptide Conjugate to Reduce Expression of a Target Gene in an Animal

[0560] In order to assess the efficiency of delivery and subsequent functionality of the dsRNAs, peptides and dsRNA-delivery peptide conjugates, subcutaneous (s.c.) tumor models (Judge et al., J Clin Invest. 2009; 119(3):661-73) are utilized. Hep3B tumors are established in female SCID/beige mice by s.c. injection of 3.times.10.sup.6 cells in 50 .mu.L PBS into the left-hind flank. Mice are randomized into treatment groups 10-17 days after seeding as tumors became palpable. Formulations of dsRNA, peptide and dsRNA-delivery peptide conjugates or PBS vehicle controls are administered by standard intravenous (i.v.) injection via the lateral tail vein, calculated based on a mg dsRNAs/kg body weight basis according to individual animal weights. Tumors are measured in 2 dimensions (width.times.length) to assess tumor growth using digital calipers. Tumor volume is calculated using the equation x*y*y/2, where x=largest diameter and y=smallest diameter, and is expressed as group mean.+-.SD. Tumor tissues are also removed from the animals of different treatment groups and gene knockdown is confirmed. Tumor volume, survival and RNA expression data are presented as a comparison between the treatments of unconjugated dsRNA versus dsRNA-delivery peptide conjugates.

Example 9

Use of a dsRNA-Targeting Peptide Conjugate to Reduce Expression of a Target Gene in a Cell

[0561] In order to assess the efficiency of targeting and subsequent functionality of the dsRNAs, peptide and dsRNA-targeting peptide conjugates, intrahepatic tumor models (Judge et al., J Clin Invest. 2009; 119(3):661-73) are used. Liver tumors are established in mice by direct intrahepatic injection of Hep3B or Neuro2a tumor cells. Female SCID/beige mice and male A/J mice are used as hosts for the Hep3B and Neuro2a tumors, respectively. Maintaining the mice under gas anesthesia, a single 1.5-cm incision across the midline is made below the sternum, and the left lateral hepatic lobe is exteriorized. 1.times.10.sup.6 Hep3B cells or 1.times.10.sup.5 Neuro2a cells suspended in 25 .mu.L PBS are injected slowly into the lobe at a shallow angle using a Hamilton syringe and a 30-gauge needle. A swab is then applied to the puncture wound to stop any bleeding prior to suturing. Mice are allowed to recover from anesthesia in a sterile cage and monitored closely for 2-4 hours before being returned to conventional housing. Eight to eleven days after tumor implantation, mice are randomized into treatment groups: dsRNA, peptide and dsRNA-peptide conjugate formulations or PBS vehicle controls are administered by standard intravenous (i.v.) injection via the lateral tail vein, calculated based on a mg dsRNAs/kg body weight basis according to individual animal weights. Body weights are monitored throughout the duration of the study as an indicator of developing tumor burden and treatment tolerability. For efficacy studies, defined humane end points are determined as a surrogate for survival. Assessments are made based on a combination of clinical signs, weight loss, and abdominal distension to define the day of euthanization due to tumor burden. Tumor tissues are removed from the animals of different treatment groups and gene knockdown is confirmed.

[0562] Functionality of peptide, dsRNA and dsRNA-peptide conjugates for tumor cell targeting are also tested by labeling the peptide and/or dsRNA with fluorescent tags and performing fluorescent biodistribution studies using a live-animal imaging system (Xenogen or BioRad) (Eguchi et al., Nat. Biotechnol. 2009 May 17. [Epub ahead of print]). Using this methodology, and by comparing with the free (i.e., unconjugated) dsRNAs the ability of the peptide to bind to the tumor cell for both the peptide alone and dsRNA-peptide conjugates is confirmed. Unconjugated dsRNAs, used as a control in this study, by contrast, are unable to bind to the same extent as conjugated dsRNAs to the tumor surface. Efficacy end points, RNA expression and biodistribution data are presented as a comparison between the treatments with unconjugated dsRNA versus dsRNA-targeting peptide conjugates.

Example 10

Use of Additional Cell Culture Models to Evaluate the Down-Regulation of KRAS Gene Expression

[0563] A variety of endpoints have been used in cell culture models to look at Ras-mediated effects after treatment with anti-Ras agents. Phenotypic endpoints include inhibition of cell proliferation, RNA expression, and reduction of Ras protein expression. Because KRAS oncogene mutations are directly associated with increased proliferation of certain tumor cells, a proliferation endpoint for cell culture assays is preferably used as the primary screen. There are several methods by which this endpoint can be measured. Following treatment of cells with DsiRNA-peptide conjugates of the invention, cells are allowed to grow (typically 5 days), after which the cell viability, the incorporation of [.sup.3H] thymidine into cellular DNA and/or the cell density are measured. The assay of cell density can be done in a 96-well format using commercially available fluorescent nucleic acid stains (such as Syto.RTM. 13 or CyQuant.RTM.). As a secondary, confirmatory endpoint, a DsiRNA-peptide-mediated decrease in the level of KRas protein expression can be evaluated using a KRas-specific ELISA.

Example 11

Evaluation of Anti-KRAS DsiRNA Efficacy in a Mouse Model of KRAS Misregulation

[0564] Anti-KRAS DsiRNA-peptide conjugates chosen from in vitro assays can be further tested in mouse models, including, e.g., xenograft and other animal models as recited above. In one example, mice possessing misregulated (e.g., elevated) KRAS levels are administered a DsiRNA-peptide agent of the present invention via hydrodynamic tail vein injection. 3-4 mice per group (divided based upon specific DsiRNA agent tested) are injected with 50 .mu.g or 200 .mu.g of DsiRNA. Levels of KRAS RNA are evaluated using RT-qPCR. Additionally or alternatively, levels of KRas (e.g., KRas protein levels and/or cancer cell/tumor formation, growth or spread) can be evaluated using an art-recognized method, or phenotypes associated with misregulation of KRAS (e.g., tumor formation, growth, metastasis, etc.) are monitored (optionally as a proxy for measurement of KRAS transcript or KRas protein levels). Active DsiRNA-peptide conjugates in such animal models can also be subsequently tested in combination with standard chemotherapies.

Example 12

Diagnostic Uses

[0565] The dsRNA-peptide molecules of the invention can be used in a variety of diagnostic applications, such as in the identification of molecular targets (e.g., RNA) in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings. Such diagnostic use of dsRNA-peptide molecules involves utilizing reconstituted RNAi systems, for example, using cellular lysates or partially purified cellular lysates. dsRNA-peptide molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells. The close relationship between dsRNA-peptide activity and the structure of the target RNA allows the detection of mutations in any region of the target molecule, which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple dsRNA-peptide molecules described in this invention, one can map nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with DsiRNA molecules can be used to inhibit gene expression and define the role of specified gene products in the progression of a disease or disorder associated with a particular target. In this manner, other genetic targets can be defined as important mediators of a disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple DsiRNA molecules targeted to different genes, DsiRNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of DsiRNA molecules and/or other chemical or biological molecules). Other in vitro uses of DsiRNA molecules of this invention are well known in the art, and include detection of the presence of RNAs associated with a disease or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a DsiRNA using standard methodologies, for example, fluorescence resonance emission transfer (FRET).

[0566] In a specific example, DsiRNA molecules that cleave only wild-type or mutant or polymorphic forms of the target KRAS RNA are used for the assay. The first DsiRNA molecules (i.e., those that cleave only wild-type forms of target KRAS RNA) are used to identify wild-type KRAS RNA present in the sample and the second DsiRNA molecules (i.e., those that cleave only mutant or polymorphic forms of target RNA) are used to identify mutant or polymorphic KRAS RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant or polymorphic KRAS RNA are cleaved by both DsiRNA molecules to demonstrate the relative DsiRNA efficiencies in the reactions and the absence of cleavage of the "non-targeted" KRAS RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant KRAS RNAs in the sample population. Thus, each analysis requires two DsiRNA molecules, two substrates and one unknown sample, which is combined into six reactions. The presence of cleavage products is determined using an RNase protection assay so that full-length and cleavage fragments of each KRAS RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant or polymorphic KRAS RNAs and putative risk of KRAS-associated phenotypic changes in target cells. The expression of KRAS mRNA whose protein product is implicated in the development of the phenotype (i.e., disease related/associated) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of KRAS RNA levels is adequate and decreases the cost of the initial diagnosis. Higher mutant or polymorphic form to wild-type ratios are correlated with higher risk whether KRAS RNA levels are compared qualitatively or quantitatively.

[0567] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

[0568] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

[0569] It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The present invention teaches one skilled in the art to test various combinations and/or substitutions of chemical modifications described herein toward generating nucleic acid constructs with improved activity for mediating RNAi activity. Such improved activity can comprise improved stability, improved bioavailability, and/or improved activation of cellular responses mediating RNAi. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying DsiRNA molecules with improved RNAi activity.

[0570] The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of", and "consisting of" may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

[0571] In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

[0572] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0573] Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.

[0574] The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Sequence CWU 1

1

161123PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 1Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr1 5 10 15Asn Lys Ala Leu Asn Asp Leu 20223PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 2Val Arg Gly Ile Ile Pro Phe Lys Thr Lys Ser Leu Asp Glu Gly Tyr1 5 10 15Asn Lys Ala Leu Asn Asp Leu 2038PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 3Lys Ser Val Lys Ala Pro Gly Ile1 5415PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 4His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn Lys Thr Leu Asp1 5 10 15512PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 5Leu Arg Leu Thr Lys Asn Ser Arg Asp Asp Ser Thr1 5 10613PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 6Lys Asn Ile Val Ser Val Lys Gly Ile Arg Lys Ser Ile1 5 10715PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 7Lys Ser Val Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu1 5 10 15813PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 8Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu Gln1 5 10918PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 9Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly His Thr Pro Gly Thr1 5 10 15Arg Leu1018PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 10Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly His Thr Ala Gly Thr1 5 10 15Arg Leu1118PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 11Glu Phe Val Met Asn Ala Ala Asn Ala Gln Gly His Thr Pro Gly Thr1 5 10 15Arg Leu1219PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 12Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg His Thr Pro Gly1 5 10 15Thr Arg Leu1321PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 13Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly His Thr1 5 10 15Pro Gly Thr Arg Leu 201422PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 14Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg His1 5 10 15Thr Pro Gly Thr Arg Leu 201527PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 15Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg Glu Asn Leu Tyr Asn Arg1 5 10 15Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu Leu 20 251624PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 16Cys Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly1 5 10 15Tyr Asn Lys Ala Leu Asn Asp Leu 201724PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 17Cys Val Arg Gly Ile Ile Pro Phe Lys Thr Lys Ser Leu Asp Glu Gly1 5 10 15Tyr Asn Lys Ala Leu Asn Asp Leu 20189PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 18Cys Lys Ser Val Lys Ala Pro Gly Ile1 51916PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 19Cys His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn Lys Thr Leu Asp1 5 10 152013PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 20Cys Leu Arg Leu Thr Lys Asn Ser Arg Asp Asp Ser Thr1 5 102114PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 21Cys Lys Asn Ile Val Ser Val Lys Gly Ile Arg Lys Ser Ile1 5 102216PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 22Cys Lys Ser Val Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu1 5 10 152314PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 23Cys Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu Gln1 5 102419PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 24Cys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly His Thr Pro Gly1 5 10 15Thr Arg Leu2519PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 25Cys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly His Thr Ala Gly1 5 10 15Thr Arg Leu2619PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 26Cys Glu Phe Val Met Asn Ala Ala Asn Ala Gln Gly His Thr Pro Gly1 5 10 15Thr Arg Leu2720PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 27Cys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg His Thr Pro1 5 10 15Gly Thr Arg Leu 202822PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 28Cys Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly His1 5 10 15Thr Pro Gly Thr Arg Leu 202923PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 29Cys Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg1 5 10 15His Thr Pro Gly Thr Arg Leu 203028PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 30Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg Glu Asn Leu Tyr Asn1 5 10 15Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu Leu 20 253124PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 31Gly Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly1 5 10 15Tyr Asn Lys Ala Leu Asn Asp Leu 203224PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 32Gly Val Arg Gly Ile Ile Pro Phe Lys Thr Lys Ser Leu Asp Glu Gly1 5 10 15Tyr Asn Lys Ala Leu Asn Asp Leu 20339PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 33Gly Lys Ser Val Lys Ala Pro Gly Ile1 53416PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 34Gly His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn Lys Thr Leu Asp1 5 10 153513PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 35Gly Leu Arg Leu Thr Lys Asn Ser Arg Asp Asp Ser Thr1 5 103614PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 36Gly Lys Asn Ile Val Ser Val Lys Gly Ile Arg Lys Ser Ile1 5 103716PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 37Gly Lys Ser Val Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu1 5 10 153814PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 38Gly Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu Gln1 5 103919PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 39Gly Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly His Thr Pro Gly1 5 10 15Thr Arg Leu4019PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 40Gly Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly His Thr Ala Gly1 5 10 15Thr Arg Leu4119PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 41Gly Glu Phe Val Met Asn Ala Ala Asn Ala Gln Gly His Thr Pro Gly1 5 10 15Thr Arg Leu4220PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 42Gly Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg His Thr Pro1 5 10 15Gly Thr Arg Leu 204322PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 43Gly Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly His1 5 10 15Thr Pro Gly Thr Arg Leu 204423PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 44Gly Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg1 5 10 15His Thr Pro Gly Thr Arg Leu 204528PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 45Gly Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg Glu Asn Leu Tyr Asn1 5 10 15Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu Leu 20 254624PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 46Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr1 5 10 15Asn Lys Ala Leu Asn Asp Leu Cys 204724PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 47Val Arg Gly Ile Ile Pro Phe Lys Thr Lys Ser Leu Asp Glu Gly Tyr1 5 10 15Asn Lys Ala Leu Asn Asp Leu Cys 20489PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 48Lys Ser Val Lys Ala Pro Gly Ile Cys1 54916PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 49His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn Lys Thr Leu Asp Cys1 5 10 155013PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 50Leu Arg Leu Thr Lys Asn Ser Arg Asp Asp Ser Thr Cys1 5 105114PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 51Lys Asn Ile Val Ser Val Lys Gly Ile Arg Lys Ser Ile Cys1 5 105216PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 52Lys Ser Val Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu Cys1 5 10 155314PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 53Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu Gln Cys1 5 105419PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 54Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly His Thr Pro Gly Thr1 5 10 15Arg Leu Cys5519PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 55Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly His Thr Ala Gly Thr1 5 10 15Arg Leu Cys5619PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 56Glu Phe Val Met Asn Ala Ala Asn Ala Gln Gly His Thr Pro Gly Thr1 5 10 15Arg Leu Cys5720PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 57Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg His Thr Pro Gly1 5 10 15Thr Arg Leu Cys 205822PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 58Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly His Thr1 5 10 15Pro Gly Thr Arg Leu Cys 205923PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 59Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg His1 5 10 15Thr Pro Gly Thr Arg Leu Cys 206028PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 60Lys Lys Ile Ile Pro Pro Thr Asn Ile Arg Glu Asn Leu Tyr Asn Arg1 5 10 15Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys 20 256118PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 61Lys Ser Val Lys Ala Pro Gly Ile Gly Gly Lys Ser Val Lys Ala Pro1 5 10 15Gly Ile6228PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 62Lys Ser Val Lys Ala Pro Gly Ile Gly Gly Lys Ser Val Lys Ala Pro1 5 10 15Gly Ile Gly Gly Lys Ser Val Lys Ala Pro Gly Ile 20 256310PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 63Lys Ser Val Lys Ala Pro Gly Ile Gly Gly1 5 106419PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 64Cys Lys Ser Val Lys Ala Pro Gly Ile Gly Gly Lys Ser Val Lys Ala1 5 10 15Pro Gly Ile6529PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 65Cys Lys Ser Val Lys Ala Pro Gly Ile Gly Gly Lys Ser Val Lys Ala1 5 10 15Pro Gly Ile Gly Gly Lys Ser Val Lys Ala Pro Gly Ile 20 256623PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 66Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Met Ile Asp Gly Trp Tyr Gly 206724PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 67Cys Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu1 5 10 15Gly Met Ile Asp Gly Trp Tyr Gly 206824PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 68Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Met Ile Asp Gly Trp Tyr Gly Cys 20696PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 69Gly Arg Gly Asp Gly Gly1 5706PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 70Cys Arg Gly Asp Gly Gly1 5716PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 71Gly Arg Gly Asp Gly Cys1 5727PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 72Thr His Ala Leu Trp His Thr1 5738PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 73Gly Thr His Ala Leu Trp His Thr1 5748PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 74Thr His Ala Leu Trp His Thr Gly1 5758PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 75Cys Thr His Ala Leu Trp His Thr1 5768PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 76Thr His Ala Leu Trp His Thr Cys1 57715PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 77Gln Pro Phe Met Gln Cys Leu Cys Leu Ile Tyr Asp Ala Ser Cys1 5 10 157816PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 78Gly Gln Pro Phe Met Gln Cys Leu Cys Leu Ile Tyr Asp Ala Ser Cys1 5 10 157916PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 79Gln Pro Phe Met Gln Cys Leu Cys Leu Ile Tyr Asp Ala Ser Cys Gly1 5 10 158015PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 80Arg Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe1 5 10 158116PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 81Gly Arg Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe1 5 10 158216PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 82Arg Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Gly1 5 10 158316PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 83Cys Arg Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe1 5 10 158416PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 84Arg Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Cys1 5 10 158514PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 85Val Phe Arg Val Arg Pro Trp Tyr Gln Ser Thr Ser Gln Ser1 5 108615PRTArtificial SequenceDescription of Artificial

Sequence Synthetic peptide 86Gly Val Phe Arg Val Arg Pro Trp Tyr Gln Ser Thr Ser Gln Ser1 5 10 158715PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 87Val Phe Arg Val Arg Pro Trp Tyr Gln Ser Thr Ser Gln Ser Gly1 5 10 158815PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 88Cys Val Phe Arg Val Arg Pro Trp Tyr Gln Ser Thr Ser Gln Ser1 5 10 158915PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 89Val Phe Arg Val Arg Pro Trp Tyr Gln Ser Thr Ser Gln Ser Cys1 5 10 1590115PRTHomo sapiens 90Met Ile Pro Ala Lys Asp Met Ala Lys Val Met Ile Val Met Leu Ala1 5 10 15Ile Cys Phe Leu Thr Lys Ser Asp Gly Lys Ser Val Lys Lys Arg Ser 20 25 30Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser 35 40 45Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn 50 55 60Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser Gln65 70 75 80Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu Lys 85 90 95Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys Ala 100 105 110Lys Ser Gln 11591138PRTHomo sapiens 91Met Thr Ala Leu Phe Leu Met Ser Met Leu Phe Gly Leu Ala Cys Gly1 5 10 15Gln Ala Met Ser Phe Cys Ile Pro Thr Glu Tyr Thr Met His Ile Glu 20 25 30Arg Arg Glu Cys Ala Tyr Cys Leu Thr Ile Asn Thr Thr Ile Cys Ala 35 40 45Gly Tyr Cys Met Thr Arg Asp Ile Asn Gly Lys Leu Phe Leu Pro Lys 50 55 60Tyr Ala Leu Ser Gln Asp Val Cys Thr Tyr Arg Asp Phe Ile Tyr Arg65 70 75 80Thr Val Glu Ile Pro Gly Cys Pro Leu His Val Ala Pro Tyr Phe Ser 85 90 95Tyr Pro Val Ala Leu Ser Cys Lys Cys Gly Lys Cys Asn Thr Asp Tyr 100 105 110Ser Asp Cys Ile His Glu Ala Ile Lys Thr Asn Tyr Cys Thr Lys Pro 115 120 125Gln Lys Ser Tyr Leu Val Gly Phe Ser Val 130 13592242PRTHomo sapiens 92Met Pro Gly Pro Trp Leu Leu Leu Ala Leu Ala Leu Thr Leu Asn Leu1 5 10 15Thr Gly Val Pro Gly Gly Arg Ala Gln Pro Glu Ala Ala Gln Gln Glu 20 25 30Ala Val Thr Ala Ala Glu His Pro Gly Leu Asp Asp Phe Leu Arg Gln 35 40 45Val Glu Arg Leu Leu Phe Leu Arg Glu Asn Ile Gln Arg Leu Gln Gly 50 55 60Asp Gln Gly Glu His Ser Ala Ser Gln Ile Phe Gln Ser Asp Trp Leu65 70 75 80Ser Lys Arg Gln His Pro Gly Lys Arg Glu Glu Glu Glu Glu Glu Gly 85 90 95Val Glu Glu Glu Glu Glu Glu Glu Gly Gly Ala Val Gly Pro His Lys 100 105 110Arg Gln His Pro Gly Arg Arg Glu Asp Glu Ala Ser Trp Ser Val Asp 115 120 125Val Thr Gln His Lys Arg Gln His Pro Gly Arg Arg Ser Pro Trp Leu 130 135 140Ala Tyr Ala Val Pro Lys Arg Gln His Pro Gly Arg Arg Leu Ala Asp145 150 155 160Pro Lys Ala Gln Arg Ser Trp Glu Glu Glu Glu Glu Glu Glu Glu Arg 165 170 175Glu Glu Asp Leu Met Pro Glu Lys Arg Gln His Pro Gly Lys Arg Ala 180 185 190Leu Gly Gly Pro Cys Gly Pro Gln Gly Ala Tyr Gly Gln Ala Gly Leu 195 200 205Leu Leu Gly Leu Leu Asp Asp Leu Ser Arg Ser Gln Gly Ala Glu Glu 210 215 220Lys Arg Gln His Pro Gly Arg Arg Ala Ala Trp Val Arg Glu Pro Leu225 230 235 240Glu Glu9392PRTHomo sapiens 93Met Lys Pro Ile Gln Lys Leu Leu Ala Gly Leu Ile Leu Leu Thr Trp1 5 10 15Cys Val Glu Gly Cys Ser Ser Gln His Trp Ser Tyr Gly Leu Arg Pro 20 25 30Gly Gly Lys Arg Asp Ala Glu Asn Leu Ile Asp Ser Phe Gln Glu Ile 35 40 45Val Lys Glu Val Gly Gln Leu Ala Glu Thr Gln Arg Phe Glu Cys Thr 50 55 60Thr His Gln Pro Arg Ser Pro Leu Arg Asp Leu Lys Gly Ala Leu Glu65 70 75 80Ser Leu Ile Glu Glu Glu Thr Gly Gln Lys Lys Ile 85 9094120PRTHomo sapiens 94Met Ala Ser Ser Arg Arg Gly Leu Leu Leu Leu Leu Leu Leu Thr Ala1 5 10 15His Leu Gly Pro Ser Glu Ala Gln His Trp Ser His Gly Trp Tyr Pro 20 25 30Gly Gly Lys Arg Ala Leu Ser Ser Ala Gln Asp Pro Gln Asn Ala Leu 35 40 45Arg Pro Pro Gly Arg Ala Leu Asp Thr Ala Ala Gly Ser Pro Val Gln 50 55 60Thr Ala His Gly Leu Pro Ser Asp Ala Leu Ala Pro Leu Asp Asp Ser65 70 75 80Met Pro Trp Glu Gly Arg Thr Thr Ala Gln Trp Ser Leu His Arg Lys 85 90 95Arg His Leu Ala Arg Thr Leu Leu Thr Ala Ala Arg Glu Pro Arg Pro 100 105 110Ala Pro Pro Ser Ser Asn Lys Val 115 12095196PRTHomo sapiens 95Met Arg Leu Pro Leu Leu Val Ser Ala Gly Val Leu Leu Val Ala Leu1 5 10 15Leu Pro Cys Pro Pro Cys Arg Ala Leu Leu Ser Arg Gly Pro Val Pro 20 25 30Gly Ala Arg Gln Ala Pro Gln His Pro Gln Pro Leu Asp Phe Phe Gln 35 40 45Pro Pro Pro Gln Ser Glu Gln Pro Gln Gln Pro Gln Ala Arg Pro Val 50 55 60Leu Leu Arg Met Gly Glu Glu Tyr Phe Leu Arg Leu Gly Asn Leu Asn65 70 75 80Lys Ser Pro Ala Ala Pro Leu Ser Pro Ala Ser Ser Leu Leu Ala Gly 85 90 95Gly Ser Gly Ser Arg Pro Ser Pro Glu Gln Ala Thr Ala Asn Phe Phe 100 105 110Arg Val Leu Leu Gln Gln Leu Leu Leu Pro Arg Arg Ser Leu Asp Ser 115 120 125Pro Ala Ala Leu Ala Glu Arg Gly Ala Arg Asn Ala Leu Gly Gly His 130 135 140Gln Glu Ala Pro Glu Arg Glu Arg Arg Ser Glu Glu Pro Pro Ile Ser145 150 155 160Leu Asp Leu Thr Phe His Leu Leu Arg Glu Val Leu Glu Met Ala Arg 165 170 175Ala Glu Gln Leu Ala Gln Gln Ala His Ser Asn Arg Lys Leu Met Glu 180 185 190Ile Ile Gly Lys 19596267PRTHomo sapiens 96Met Pro Arg Ser Cys Cys Ser Arg Ser Gly Ala Leu Leu Leu Ala Leu1 5 10 15Leu Leu Gln Ala Ser Met Glu Val Arg Gly Trp Cys Leu Glu Ser Ser 20 25 30Gln Cys Gln Asp Leu Thr Thr Glu Ser Asn Leu Leu Glu Cys Ile Arg 35 40 45Ala Cys Lys Pro Asp Leu Ser Ala Glu Thr Pro Met Phe Pro Gly Asn 50 55 60Gly Asp Glu Gln Pro Leu Thr Glu Asn Pro Arg Lys Tyr Val Met Gly65 70 75 80His Phe Arg Trp Asp Arg Phe Gly Arg Arg Asn Ser Ser Ser Ser Gly 85 90 95Ser Ser Gly Ala Gly Gln Lys Arg Glu Asp Val Ser Ala Gly Glu Asp 100 105 110Cys Gly Pro Leu Pro Glu Gly Gly Pro Glu Pro Arg Ser Asp Gly Ala 115 120 125Lys Pro Gly Pro Arg Glu Gly Lys Arg Ser Tyr Ser Met Glu His Phe 130 135 140Arg Trp Gly Lys Pro Val Gly Lys Lys Arg Arg Pro Val Lys Val Tyr145 150 155 160Pro Asn Gly Ala Glu Asp Glu Ser Ala Glu Ala Phe Pro Leu Glu Phe 165 170 175Lys Arg Glu Leu Thr Gly Gln Arg Leu Arg Glu Gly Asp Gly Pro Asp 180 185 190Gly Pro Ala Asp Asp Gly Ala Gly Ala Gln Ala Asp Leu Glu His Ser 195 200 205Leu Leu Val Ala Ala Glu Lys Lys Asp Glu Gly Pro Tyr Arg Met Glu 210 215 220His Phe Arg Trp Gly Ser Pro Pro Lys Asp Lys Arg Tyr Gly Gly Phe225 230 235 240Met Thr Ser Glu Lys Ser Gln Thr Pro Leu Val Thr Leu Phe Lys Asn 245 250 255Ala Ile Ile Lys Asn Ala Tyr Lys Lys Gly Glu 260 26597622PRTHomo sapiens 97Met Ala His Val Arg Gly Leu Gln Leu Pro Gly Cys Leu Ala Leu Ala1 5 10 15Ala Leu Cys Ser Leu Val His Ser Gln His Val Phe Leu Ala Pro Gln 20 25 30Gln Ala Arg Ser Leu Leu Gln Arg Val Arg Arg Ala Asn Thr Phe Leu 35 40 45Glu Glu Val Arg Lys Gly Asn Leu Glu Arg Glu Cys Val Glu Glu Thr 50 55 60Cys Ser Tyr Glu Glu Ala Phe Glu Ala Leu Glu Ser Ser Thr Ala Thr65 70 75 80Asp Val Phe Trp Ala Lys Tyr Thr Ala Cys Glu Thr Ala Arg Thr Pro 85 90 95Arg Asp Lys Leu Ala Ala Cys Leu Glu Gly Asn Cys Ala Glu Gly Leu 100 105 110Gly Thr Asn Tyr Arg Gly His Val Asn Ile Thr Arg Ser Gly Ile Glu 115 120 125Cys Gln Leu Trp Arg Ser Arg Tyr Pro His Lys Pro Glu Ile Asn Ser 130 135 140Thr Thr His Pro Gly Ala Asp Leu Gln Glu Asn Phe Cys Arg Asn Pro145 150 155 160Asp Ser Ser Thr Thr Gly Pro Trp Cys Tyr Thr Thr Asp Pro Thr Val 165 170 175Arg Arg Gln Glu Cys Ser Ile Pro Val Cys Gly Gln Asp Gln Val Thr 180 185 190Val Ala Met Thr Pro Arg Ser Glu Gly Ser Ser Val Asn Leu Ser Pro 195 200 205Pro Leu Glu Gln Cys Val Pro Asp Arg Gly Gln Gln Tyr Gln Gly Arg 210 215 220Leu Ala Val Thr Thr His Gly Leu Pro Cys Leu Ala Trp Ala Ser Ala225 230 235 240Gln Ala Lys Ala Leu Ser Lys His Gln Asp Phe Asn Ser Ala Val Gln 245 250 255Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Gly Asp Glu Glu Gly Val 260 265 270Trp Cys Tyr Val Ala Gly Lys Pro Gly Asp Phe Gly Tyr Cys Asp Leu 275 280 285Asn Tyr Cys Glu Glu Ala Val Glu Glu Glu Thr Gly Asp Gly Leu Asp 290 295 300Glu Asp Ser Asp Arg Ala Ile Glu Gly Arg Thr Ala Thr Ser Glu Tyr305 310 315 320Gln Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser Gly Glu Ala Asp Cys 325 330 335Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu Glu Asp Lys Thr Glu 340 345 350Arg Glu Leu Leu Glu Ser Tyr Ile Asp Gly Arg Ile Val Glu Gly Ser 355 360 365Asp Ala Glu Ile Gly Met Ser Pro Trp Gln Val Met Leu Phe Arg Lys 370 375 380Ser Pro Gln Glu Leu Leu Cys Gly Ala Ser Leu Ile Ser Asp Arg Trp385 390 395 400Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro Pro Trp Asp Lys Asn 405 410 415Phe Thr Glu Asn Asp Leu Leu Val Arg Ile Gly Lys His Ser Arg Thr 420 425 430Arg Tyr Glu Arg Asn Ile Glu Lys Ile Ser Met Leu Glu Lys Ile Tyr 435 440 445Ile His Pro Arg Tyr Asn Trp Arg Glu Asn Leu Asp Arg Asp Ile Ala 450 455 460Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser Asp Tyr Ile His Pro465 470 475 480Val Cys Leu Pro Asp Arg Glu Thr Ala Ala Ser Leu Leu Gln Ala Gly 485 490 495Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Leu Lys Glu Thr Trp Thr 500 505 510Ala Asn Val Gly Lys Gly Gln Pro Ser Val Leu Gln Val Val Asn Leu 515 520 525Pro Ile Val Glu Arg Pro Val Cys Lys Asp Ser Thr Arg Ile Arg Ile 530 535 540Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro Asp Glu Gly Lys Arg545 550 555 560Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe Val Met Lys Ser 565 570 575Pro Phe Asn Asn Arg Trp Tyr Gln Met Gly Ile Val Ser Trp Gly Glu 580 585 590Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr Thr His Val Phe Arg 595 600 605Leu Lys Lys Trp Ile Gln Lys Val Ile Asp Gln Phe Gly Glu 610 615 620981663PRTHomo sapiens 98Met Gly Pro Thr Ser Gly Pro Ser Leu Leu Leu Leu Leu Leu Thr His1 5 10 15Leu Pro Leu Ala Leu Gly Ser Pro Met Tyr Ser Ile Ile Thr Pro Asn 20 25 30Ile Leu Arg Leu Glu Ser Glu Glu Thr Met Val Leu Glu Ala His Asp 35 40 45Ala Gln Gly Asp Val Pro Val Thr Val Thr Val His Asp Phe Pro Gly 50 55 60Lys Lys Leu Val Leu Ser Ser Glu Lys Thr Val Leu Thr Pro Ala Thr65 70 75 80Asn His Met Gly Asn Val Thr Phe Thr Ile Pro Ala Asn Arg Glu Phe 85 90 95Lys Ser Glu Lys Gly Arg Asn Lys Phe Val Thr Val Gln Ala Thr Phe 100 105 110Gly Thr Gln Val Val Glu Lys Val Val Leu Val Ser Leu Gln Ser Gly 115 120 125Tyr Leu Phe Ile Gln Thr Asp Lys Thr Ile Tyr Thr Pro Gly Ser Thr 130 135 140Val Leu Tyr Arg Ile Phe Thr Val Asn His Lys Leu Leu Pro Val Gly145 150 155 160Arg Thr Val Met Val Asn Ile Glu Asn Pro Glu Gly Ile Pro Val Lys 165 170 175Gln Asp Ser Leu Ser Ser Gln Asn Gln Leu Gly Val Leu Pro Leu Ser 180 185 190Trp Asp Ile Pro Glu Leu Val Asn Met Gly Gln Trp Lys Ile Arg Ala 195 200 205Tyr Tyr Glu Asn Ser Pro Gln Gln Val Phe Ser Thr Glu Phe Glu Val 210 215 220Lys Glu Tyr Val Leu Pro Ser Phe Glu Val Ile Val Glu Pro Thr Glu225 230 235 240Lys Phe Tyr Tyr Ile Tyr Asn Glu Lys Gly Leu Glu Val Thr Ile Thr 245 250 255Ala Arg Phe Leu Tyr Gly Lys Lys Val Glu Gly Thr Ala Phe Val Ile 260 265 270Phe Gly Ile Gln Asp Gly Glu Gln Arg Ile Ser Leu Pro Glu Ser Leu 275 280 285Lys Arg Ile Pro Ile Glu Asp Gly Ser Gly Glu Val Val Leu Ser Arg 290 295 300Lys Val Leu Leu Asp Gly Val Gln Asn Pro Arg Ala Glu Asp Leu Val305 310 315 320Gly Lys Ser Leu Tyr Val Ser Ala Thr Val Ile Leu His Ser Gly Ser 325 330 335Asp Met Val Gln Ala Glu Arg Ser Gly Ile Pro Ile Val Thr Ser Pro 340 345 350Tyr Gln Ile His Phe Thr Lys Thr Pro Lys Tyr Phe Lys Pro Gly Met 355 360 365Pro Phe Asp Leu Met Val Phe Val Thr Asn Pro Asp Gly Ser Pro Ala 370 375 380Tyr Arg Val Pro Val Ala Val Gln Gly Glu Asp Thr Val Gln Ser Leu385 390 395 400Thr Gln Gly Asp Gly Val Ala Lys Leu Ser Ile Asn Thr His Pro Ser 405 410 415Gln Lys Pro Leu Ser Ile Thr Val Arg Thr Lys Lys Gln Glu Leu Ser 420 425 430Glu Ala Glu Gln Ala Thr Arg Thr Met Gln Ala Leu Pro Tyr Ser Thr 435 440 445Val Gly Asn Ser Asn Asn Tyr Leu His Leu Ser Val Leu Arg Thr Glu 450 455 460Leu Arg Pro Gly Glu Thr Leu Asn Val Asn Phe Leu Leu Arg Met Asp465 470 475 480Arg Ala His Glu Ala Lys Ile Arg Tyr Tyr Thr Tyr Leu Ile Met Asn 485 490 495Lys Gly Arg Leu Leu Lys Ala Gly Arg Gln Val Arg Glu Pro Gly Gln 500 505 510Asp Leu Val Val Leu Pro Leu Ser Ile Thr Thr Asp Phe Ile Pro Ser 515 520 525Phe Arg Leu Val Ala Tyr Tyr Thr Leu Ile Gly Ala Ser Gly Gln Arg 530 535 540Glu Val Val Ala Asp Ser Val Trp Val Asp Val Lys Asp Ser Cys Val545 550

555 560Gly Ser Leu Val Val Lys Ser Gly Gln Ser Glu Asp Arg Gln Pro Val 565 570 575Pro Gly Gln Gln Met Thr Leu Lys Ile Glu Gly Asp His Gly Ala Arg 580 585 590Val Val Leu Val Ala Val Asp Lys Gly Val Phe Val Leu Asn Lys Lys 595 600 605Asn Lys Leu Thr Gln Ser Lys Ile Trp Asp Val Val Glu Lys Ala Asp 610 615 620Ile Gly Cys Thr Pro Gly Ser Gly Lys Asp Tyr Ala Gly Val Phe Ser625 630 635 640Asp Ala Gly Leu Thr Phe Thr Ser Ser Ser Gly Gln Gln Thr Ala Gln 645 650 655Arg Ala Glu Leu Gln Cys Pro Gln Pro Ala Ala Arg Arg Arg Arg Ser 660 665 670Val Gln Leu Thr Glu Lys Arg Met Asp Lys Val Gly Lys Tyr Pro Lys 675 680 685Glu Leu Arg Lys Cys Cys Glu Asp Gly Met Arg Glu Asn Pro Met Arg 690 695 700Phe Ser Cys Gln Arg Arg Thr Arg Phe Ile Ser Leu Gly Glu Ala Cys705 710 715 720Lys Lys Val Phe Leu Asp Cys Cys Asn Tyr Ile Thr Glu Leu Arg Arg 725 730 735Gln His Ala Arg Ala Ser His Leu Gly Leu Ala Arg Ser Asn Leu Asp 740 745 750Glu Asp Ile Ile Ala Glu Glu Asn Ile Val Ser Arg Ser Glu Phe Pro 755 760 765Glu Ser Trp Leu Trp Asn Val Glu Asp Leu Lys Glu Pro Pro Lys Asn 770 775 780Gly Ile Ser Thr Lys Leu Met Asn Ile Phe Leu Lys Asp Ser Ile Thr785 790 795 800Thr Trp Glu Ile Leu Ala Val Ser Met Ser Asp Lys Lys Gly Ile Cys 805 810 815Val Ala Asp Pro Phe Glu Val Thr Val Met Gln Asp Phe Phe Ile Asp 820 825 830Leu Arg Leu Pro Tyr Ser Val Val Arg Asn Glu Gln Val Glu Ile Arg 835 840 845Ala Val Leu Tyr Asn Tyr Arg Gln Asn Gln Glu Leu Lys Val Arg Val 850 855 860Glu Leu Leu His Asn Pro Ala Phe Cys Ser Leu Ala Thr Thr Lys Arg865 870 875 880Arg His Gln Gln Thr Val Thr Ile Pro Pro Lys Ser Ser Leu Ser Val 885 890 895Pro Tyr Val Ile Val Pro Leu Lys Thr Gly Leu Gln Glu Val Glu Val 900 905 910Lys Ala Ala Val Tyr His His Phe Ile Ser Asp Gly Val Arg Lys Ser 915 920 925Leu Lys Val Val Pro Glu Gly Ile Arg Met Asn Lys Thr Val Ala Val 930 935 940Arg Thr Leu Asp Pro Glu Arg Leu Gly Arg Glu Gly Val Gln Lys Glu945 950 955 960Asp Ile Pro Pro Ala Asp Leu Ser Asp Gln Val Pro Asp Thr Glu Ser 965 970 975Glu Thr Arg Ile Leu Leu Gln Gly Thr Pro Val Ala Gln Met Thr Glu 980 985 990Asp Ala Val Asp Ala Glu Arg Leu Lys His Leu Ile Val Thr Pro Ser 995 1000 1005Gly Cys Gly Glu Gln Asn Met Ile Gly Met Thr Pro Thr Val Ile 1010 1015 1020Ala Val His Tyr Leu Asp Glu Thr Glu Gln Trp Glu Lys Phe Gly 1025 1030 1035Leu Glu Lys Arg Gln Gly Ala Leu Glu Leu Ile Lys Lys Gly Tyr 1040 1045 1050Thr Gln Gln Leu Ala Phe Arg Gln Pro Ser Ser Ala Phe Ala Ala 1055 1060 1065Phe Val Lys Arg Ala Pro Ser Thr Trp Leu Thr Ala Tyr Val Val 1070 1075 1080Lys Val Phe Ser Leu Ala Val Asn Leu Ile Ala Ile Asp Ser Gln 1085 1090 1095Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu Glu Lys Gln Lys 1100 1105 1110Pro Asp Gly Val Phe Gln Glu Asp Ala Pro Val Ile His Gln Glu 1115 1120 1125Met Ile Gly Gly Leu Arg Asn Asn Asn Glu Lys Asp Met Ala Leu 1130 1135 1140Thr Ala Phe Val Leu Ile Ser Leu Gln Glu Ala Lys Asp Ile Cys 1145 1150 1155Glu Glu Gln Val Asn Ser Leu Pro Gly Ser Ile Thr Lys Ala Gly 1160 1165 1170Asp Phe Leu Glu Ala Asn Tyr Met Asn Leu Gln Arg Ser Tyr Thr 1175 1180 1185Val Ala Ile Ala Gly Tyr Ala Leu Ala Gln Met Gly Arg Leu Lys 1190 1195 1200Gly Pro Leu Leu Asn Lys Phe Leu Thr Thr Ala Lys Asp Lys Asn 1205 1210 1215Arg Trp Glu Asp Pro Gly Lys Gln Leu Tyr Asn Val Glu Ala Thr 1220 1225 1230Ser Tyr Ala Leu Leu Ala Leu Leu Gln Leu Lys Asp Phe Asp Phe 1235 1240 1245Val Pro Pro Val Val Arg Trp Leu Asn Glu Gln Arg Tyr Tyr Gly 1250 1255 1260Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe Met Val Phe Gln Ala 1265 1270 1275Leu Ala Gln Tyr Gln Lys Asp Ala Pro Asp His Gln Glu Leu Asn 1280 1285 1290Leu Asp Val Ser Leu Gln Leu Pro Ser Arg Ser Ser Lys Ile Thr 1295 1300 1305His Arg Ile His Trp Glu Ser Ala Ser Leu Leu Arg Ser Glu Glu 1310 1315 1320Thr Lys Glu Asn Glu Gly Phe Thr Val Thr Ala Glu Gly Lys Gly 1325 1330 1335Gln Gly Thr Leu Ser Val Val Thr Met Tyr His Ala Lys Ala Lys 1340 1345 1350Asp Gln Leu Thr Cys Asn Lys Phe Asp Leu Lys Val Thr Ile Lys 1355 1360 1365Pro Ala Pro Glu Thr Glu Lys Arg Pro Gln Asp Ala Lys Asn Thr 1370 1375 1380Met Ile Leu Glu Ile Cys Thr Arg Tyr Arg Gly Asp Gln Asp Ala 1385 1390 1395Thr Met Ser Ile Leu Asp Ile Ser Met Met Thr Gly Phe Ala Pro 1400 1405 1410Asp Thr Asp Asp Leu Lys Gln Leu Ala Asn Gly Val Asp Arg Tyr 1415 1420 1425Ile Ser Lys Tyr Glu Leu Asp Lys Ala Phe Ser Asp Arg Asn Thr 1430 1435 1440Leu Ile Ile Tyr Leu Asp Lys Val Ser His Ser Glu Asp Asp Cys 1445 1450 1455Leu Ala Phe Lys Val His Gln Tyr Phe Asn Val Glu Leu Ile Gln 1460 1465 1470Pro Gly Ala Val Lys Val Tyr Ala Tyr Tyr Asn Leu Glu Glu Ser 1475 1480 1485Cys Thr Arg Phe Tyr His Pro Glu Lys Glu Asp Gly Lys Leu Asn 1490 1495 1500Lys Leu Cys Arg Asp Glu Leu Cys Arg Cys Ala Glu Glu Asn Cys 1505 1510 1515Phe Ile Gln Lys Ser Asp Asp Lys Val Thr Leu Glu Glu Arg Leu 1520 1525 1530Asp Lys Ala Cys Glu Pro Gly Val Asp Tyr Val Tyr Lys Thr Arg 1535 1540 1545Leu Val Lys Val Gln Leu Ser Asn Asp Phe Asp Glu Tyr Ile Met 1550 1555 1560Ala Ile Glu Gln Thr Ile Lys Ser Gly Ser Asp Glu Val Gln Val 1565 1570 1575Gly Gln Gln Arg Thr Phe Ile Ser Pro Ile Lys Cys Arg Glu Ala 1580 1585 1590Leu Lys Leu Glu Glu Lys Lys His Tyr Leu Met Trp Gly Leu Ser 1595 1600 1605Ser Asp Phe Trp Gly Glu Lys Pro Asn Leu Ser Tyr Ile Ile Gly 1610 1615 1620Lys Asp Thr Trp Val Glu His Trp Pro Glu Glu Asp Glu Cys Gln 1625 1630 1635Asp Glu Glu Asn Gln Lys Gln Cys Gln Asp Leu Gly Ala Phe Thr 1640 1645 1650Glu Ser Met Val Val Phe Gly Cys Pro Asn 1655 1660991744PRTHomo sapiens 99Met Arg Leu Leu Trp Gly Leu Ile Trp Ala Ser Ser Phe Phe Thr Leu1 5 10 15Ser Leu Gln Lys Pro Arg Leu Leu Leu Phe Ser Pro Ser Val Val His 20 25 30Leu Gly Val Pro Leu Ser Val Gly Val Gln Leu Gln Asp Val Pro Arg 35 40 45Gly Gln Val Val Lys Gly Ser Val Phe Leu Arg Asn Pro Ser Arg Asn 50 55 60Asn Val Pro Cys Ser Pro Lys Val Asp Phe Thr Leu Ser Ser Glu Arg65 70 75 80Asp Phe Ala Leu Leu Ser Leu Gln Val Pro Leu Lys Asp Ala Lys Ser 85 90 95Cys Gly Leu His Gln Leu Leu Arg Gly Pro Glu Val Gln Leu Val Ala 100 105 110His Ser Pro Trp Leu Lys Asp Ser Leu Ser Arg Thr Thr Asn Ile Gln 115 120 125Gly Ile Asn Leu Leu Phe Ser Ser Arg Arg Gly His Leu Phe Leu Gln 130 135 140Thr Asp Gln Pro Ile Tyr Asn Pro Gly Gln Arg Val Arg Tyr Arg Val145 150 155 160Phe Ala Leu Asp Gln Lys Met Arg Pro Ser Thr Asp Thr Ile Thr Val 165 170 175Met Val Glu Asn Ser His Gly Leu Arg Val Arg Lys Lys Glu Val Tyr 180 185 190Met Pro Ser Ser Ile Phe Gln Asp Asp Phe Val Ile Pro Asp Ile Ser 195 200 205Glu Pro Gly Thr Trp Lys Ile Ser Ala Arg Phe Ser Asp Gly Leu Glu 210 215 220Ser Asn Ser Ser Thr Gln Phe Glu Val Lys Lys Tyr Val Leu Pro Asn225 230 235 240Phe Glu Val Lys Ile Thr Pro Gly Lys Pro Tyr Ile Leu Thr Val Pro 245 250 255Gly His Leu Asp Glu Met Gln Leu Asp Ile Gln Ala Arg Tyr Ile Tyr 260 265 270Gly Lys Pro Val Gln Gly Val Ala Tyr Val Arg Phe Gly Leu Leu Asp 275 280 285Glu Asp Gly Lys Lys Thr Phe Phe Arg Gly Leu Glu Ser Gln Thr Lys 290 295 300Leu Val Asn Gly Gln Ser His Ile Ser Leu Ser Lys Ala Glu Phe Gln305 310 315 320Asp Ala Leu Glu Lys Leu Asn Met Gly Ile Thr Asp Leu Gln Gly Leu 325 330 335Arg Leu Tyr Val Ala Ala Ala Ile Ile Glu Ser Pro Gly Gly Glu Met 340 345 350Glu Glu Ala Glu Leu Thr Ser Trp Tyr Phe Val Ser Ser Pro Phe Ser 355 360 365Leu Asp Leu Ser Lys Thr Lys Arg His Leu Val Pro Gly Ala Pro Phe 370 375 380Leu Leu Gln Ala Leu Val Arg Glu Met Ser Gly Ser Pro Ala Ser Gly385 390 395 400Ile Pro Val Lys Val Ser Ala Thr Val Ser Ser Pro Gly Ser Val Pro 405 410 415Glu Val Gln Asp Ile Gln Gln Asn Thr Asp Gly Ser Gly Gln Val Ser 420 425 430Ile Pro Ile Ile Ile Pro Gln Thr Ile Ser Glu Leu Gln Leu Ser Val 435 440 445Ser Ala Gly Ser Pro His Pro Ala Ile Ala Arg Leu Thr Val Ala Ala 450 455 460Pro Pro Ser Gly Gly Pro Gly Phe Leu Ser Ile Glu Arg Pro Asp Ser465 470 475 480Arg Pro Pro Arg Val Gly Asp Thr Leu Asn Leu Asn Leu Arg Ala Val 485 490 495Gly Ser Gly Ala Thr Phe Ser His Tyr Tyr Tyr Met Ile Leu Ser Arg 500 505 510Gly Gln Ile Val Phe Met Asn Arg Glu Pro Lys Arg Thr Leu Thr Ser 515 520 525Val Ser Val Phe Val Asp His His Leu Ala Pro Ser Phe Tyr Phe Val 530 535 540Ala Phe Tyr Tyr His Gly Asp His Pro Val Ala Asn Ser Leu Arg Val545 550 555 560Asp Val Gln Ala Gly Ala Cys Glu Gly Lys Leu Glu Leu Ser Val Asp 565 570 575Gly Ala Lys Gln Tyr Arg Asn Gly Glu Ser Val Lys Leu His Leu Glu 580 585 590Thr Asp Ser Leu Ala Leu Val Ala Leu Gly Ala Leu Asp Thr Ala Leu 595 600 605Tyr Ala Ala Gly Ser Lys Ser His Lys Pro Leu Asn Met Gly Lys Val 610 615 620Phe Glu Ala Met Asn Ser Tyr Asp Leu Gly Cys Gly Pro Gly Gly Gly625 630 635 640Asp Ser Ala Leu Gln Val Phe Gln Ala Ala Gly Leu Ala Phe Ser Asp 645 650 655Gly Asp Gln Trp Thr Leu Ser Arg Lys Arg Leu Ser Cys Pro Lys Glu 660 665 670Lys Thr Thr Arg Lys Lys Arg Asn Val Asn Phe Gln Lys Ala Ile Asn 675 680 685Glu Lys Leu Gly Gln Tyr Ala Ser Pro Thr Ala Lys Arg Cys Cys Gln 690 695 700Asp Gly Val Thr Arg Leu Pro Met Met Arg Ser Cys Glu Gln Arg Ala705 710 715 720Ala Arg Val Gln Gln Pro Asp Cys Arg Glu Pro Phe Leu Ser Cys Cys 725 730 735Gln Phe Ala Glu Ser Leu Arg Lys Lys Ser Arg Asp Lys Gly Gln Ala 740 745 750Gly Leu Gln Arg Ala Leu Glu Ile Leu Gln Glu Glu Asp Leu Ile Asp 755 760 765Glu Asp Asp Ile Pro Val Arg Ser Phe Phe Pro Glu Asn Trp Leu Trp 770 775 780Arg Val Glu Thr Val Asp Arg Phe Gln Ile Leu Thr Leu Trp Leu Pro785 790 795 800Asp Ser Leu Thr Thr Trp Glu Ile His Gly Leu Ser Leu Ser Lys Thr 805 810 815Lys Gly Leu Cys Val Ala Thr Pro Val Gln Leu Arg Val Phe Arg Glu 820 825 830Phe His Leu His Leu Arg Leu Pro Met Ser Val Arg Arg Phe Glu Gln 835 840 845Leu Glu Leu Arg Pro Val Leu Tyr Asn Tyr Leu Asp Lys Asn Leu Thr 850 855 860Val Ser Val His Val Ser Pro Val Glu Gly Leu Cys Leu Ala Gly Gly865 870 875 880Gly Gly Leu Ala Gln Gln Val Leu Val Pro Ala Gly Ser Ala Arg Pro 885 890 895Val Ala Phe Ser Val Val Pro Thr Ala Ala Ala Ala Val Ser Leu Lys 900 905 910Val Val Ala Arg Gly Ser Phe Glu Phe Pro Val Gly Asp Ala Val Ser 915 920 925Lys Val Leu Gln Ile Glu Lys Glu Gly Ala Ile His Arg Glu Glu Leu 930 935 940Val Tyr Glu Leu Asn Pro Leu Asp His Arg Gly Arg Thr Leu Glu Ile945 950 955 960Pro Gly Asn Ser Asp Pro Asn Met Ile Pro Asp Gly Asp Phe Asn Ser 965 970 975Tyr Val Arg Val Thr Ala Ser Asp Pro Leu Asp Thr Leu Gly Ser Glu 980 985 990Gly Ala Leu Ser Pro Gly Gly Val Ala Ser Leu Leu Arg Leu Pro Arg 995 1000 1005Gly Cys Gly Glu Gln Thr Met Ile Tyr Leu Ala Pro Thr Leu Ala 1010 1015 1020Ala Ser Arg Tyr Leu Asp Lys Thr Glu Gln Trp Ser Thr Leu Pro 1025 1030 1035Pro Glu Thr Lys Asp His Ala Val Asp Leu Ile Gln Lys Gly Tyr 1040 1045 1050Met Arg Ile Gln Gln Phe Arg Lys Ala Asp Gly Ser Tyr Ala Ala 1055 1060 1065Trp Leu Ser Arg Asp Ser Ser Thr Trp Leu Thr Ala Phe Val Leu 1070 1075 1080Lys Val Leu Ser Leu Ala Gln Glu Gln Val Gly Gly Ser Pro Glu 1085 1090 1095Lys Leu Gln Glu Thr Ser Asn Trp Leu Leu Ser Gln Gln Gln Ala 1100 1105 1110Asp Gly Ser Phe Gln Asp Leu Ser Pro Val Ile His Arg Ser Met 1115 1120 1125Gln Gly Gly Leu Val Gly Asn Asp Glu Thr Val Ala Leu Thr Ala 1130 1135 1140Phe Val Thr Ile Ala Leu His His Gly Leu Ala Val Phe Gln Asp 1145 1150 1155Glu Gly Ala Glu Pro Leu Lys Gln Arg Val Glu Ala Ser Ile Ser 1160 1165 1170Lys Ala Asn Ser Phe Leu Gly Glu Lys Ala Ser Ala Gly Leu Leu 1175 1180 1185Gly Ala His Ala Ala Ala Ile Thr Ala Tyr Ala Leu Ser Leu Thr 1190 1195 1200Lys Ala Pro Val Asp Leu Leu Gly Val Ala His Asn Asn Leu Met 1205 1210 1215Ala Met Ala Gln Glu Thr Gly Asp Asn Leu Tyr Trp Gly Ser Val 1220 1225 1230Thr Gly Ser Gln Ser Asn Ala Val Ser Pro Thr Pro Ala Pro Arg 1235 1240 1245Asn Pro Ser Asp Pro Met Pro Gln Ala Pro Ala Leu Trp Ile Glu 1250 1255 1260Thr Thr Ala Tyr Ala Leu Leu His Leu Leu Leu His Glu Gly Lys 1265 1270 1275Ala Glu Met Ala Asp Gln Ala Ser Ala Trp Leu Thr Arg Gln Gly 1280 1285 1290Ser Phe Gln Gly Gly Phe Arg Ser Thr Gln Asp Thr Val Ile Ala 1295 1300 1305Leu Asp Ala Leu Ser Ala Tyr Trp Ile Ala Ser His Thr Thr Glu 1310 1315 1320Glu Arg Gly Leu Asn Val Thr Leu Ser Ser Thr Gly Arg Asn Gly 1325 1330 1335Phe Lys Ser His Ala Leu Gln Leu Asn Asn Arg Gln Ile Arg Gly

1340 1345 1350Leu Glu Glu Glu Leu Gln Phe Ser Leu Gly Ser Lys Ile Asn Val 1355 1360 1365Lys Val Gly Gly Asn Ser Lys Gly Thr Leu Lys Val Leu Arg Thr 1370 1375 1380Tyr Asn Val Leu Asp Met Lys Asn Thr Thr Cys Gln Asp Leu Gln 1385 1390 1395Ile Glu Val Thr Val Lys Gly His Val Glu Tyr Thr Met Glu Ala 1400 1405 1410Asn Glu Asp Tyr Glu Asp Tyr Glu Tyr Asp Glu Leu Pro Ala Lys 1415 1420 1425Asp Asp Pro Asp Ala Pro Leu Gln Pro Val Thr Pro Leu Gln Leu 1430 1435 1440Phe Glu Gly Arg Arg Asn Arg Arg Arg Arg Glu Ala Pro Lys Val 1445 1450 1455Val Glu Glu Gln Glu Ser Arg Val His Tyr Thr Val Cys Ile Trp 1460 1465 1470Arg Asn Gly Lys Val Gly Leu Ser Gly Met Ala Ile Ala Asp Val 1475 1480 1485Thr Leu Leu Ser Gly Phe His Ala Leu Arg Ala Asp Leu Glu Lys 1490 1495 1500Leu Thr Ser Leu Ser Asp Arg Tyr Val Ser His Phe Glu Thr Glu 1505 1510 1515Gly Pro His Val Leu Leu Tyr Phe Asp Ser Val Pro Thr Ser Arg 1520 1525 1530Glu Cys Val Gly Phe Glu Ala Val Gln Glu Val Pro Val Gly Leu 1535 1540 1545Val Gln Pro Ala Ser Ala Thr Leu Tyr Asp Tyr Tyr Asn Pro Glu 1550 1555 1560Arg Arg Cys Ser Val Phe Tyr Gly Ala Pro Ser Lys Ser Arg Leu 1565 1570 1575Leu Ala Thr Leu Cys Ser Ala Glu Val Cys Gln Cys Ala Glu Gly 1580 1585 1590Lys Cys Pro Arg Gln Arg Arg Ala Leu Glu Arg Gly Leu Gln Asp 1595 1600 1605Glu Asp Gly Tyr Arg Met Lys Phe Ala Cys Tyr Tyr Pro Arg Val 1610 1615 1620Glu Tyr Gly Phe Gln Val Lys Val Leu Arg Glu Asp Ser Arg Ala 1625 1630 1635Ala Phe Arg Leu Phe Glu Thr Lys Ile Thr Gln Val Leu His Phe 1640 1645 1650Thr Lys Asp Val Lys Ala Ala Ala Asn Gln Met Arg Asn Phe Leu 1655 1660 1665Val Arg Ala Ser Cys Arg Leu Arg Leu Glu Pro Gly Lys Glu Tyr 1670 1675 1680Leu Ile Met Gly Leu Asp Gly Ala Thr Tyr Asp Leu Glu Gly His 1685 1690 1695Pro Gln Tyr Leu Leu Asp Ser Asn Ser Trp Ile Glu Glu Met Pro 1700 1705 1710Ser Glu Arg Leu Cys Arg Ser Thr Arg Gln Arg Ala Ala Cys Ala 1715 1720 1725Gln Leu Asn Asp Phe Leu Gln Glu Tyr Gly Thr Gln Gly Cys Gln 1730 1735 1740Val100770PRTHomo sapiens 100Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg1 5 10 15Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35 40 45Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu65 70 75 80Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val 100 105 110Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys 130 135 140Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu145 150 155 160Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170 175Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu225 230 235 240Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290 295 300Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe305 310 315 320Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr 340 345 350Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala 355 360 365Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp 370 375 380Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala385 390 395 400Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala 405 410 415Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile 420 425 430Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn 435 440 445Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met 450 455 460Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu465 470 475 480Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys 485 490 495Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe 500 505 510Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser 515 520 525Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser 530 535 540Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp545 550 555 560Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val 565 570 575Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala 580 585 590Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro 595 600 605Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe 610 615 620Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val625 630 635 640Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser 645 650 655Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp 660 665 670Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu 675 680 685Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly 690 695 700Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu705 710 715 720Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val 725 730 735Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met 740 745 750Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met 755 760 765Gln Asn 770101217PRTHomo sapiens 101Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu1 5 10 15Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu 20 25 30Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln 35 40 45Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys 50 55 60Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe65 70 75 80Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys 85 90 95Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp 100 105 110Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val 115 120 125Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu 130 135 140Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg145 150 155 160Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser 165 170 175His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe 180 185 190Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys 195 200 205Arg Ser Val Glu Gly Ser Cys Gly Phe 210 215102117PRTHomo sapiens 102Met Pro Ser Pro Gly Thr Val Cys Ser Leu Leu Leu Leu Gly Met Leu1 5 10 15Trp Leu Asp Leu Ala Met Ala Gly Ser Ser Phe Leu Ser Pro Glu His 20 25 30Gln Arg Val Gln Gln Arg Lys Glu Ser Lys Lys Pro Pro Ala Lys Leu 35 40 45Gln Pro Arg Ala Leu Ala Gly Trp Leu Arg Pro Glu Asp Gly Gly Gln 50 55 60Ala Glu Gly Ala Glu Asp Glu Leu Glu Val Arg Phe Asn Ala Pro Phe65 70 75 80Asp Val Gly Ile Lys Leu Ser Gly Val Gln Tyr Gln Gln His Ser Gln 85 90 95Ala Leu Gly Lys Phe Leu Gln Asp Ile Leu Trp Glu Glu Ala Lys Glu 100 105 110Ala Pro Ala Asp Lys 115103390PRTHomo sapiens 103Met Pro Pro Ser Gly Leu Arg Leu Leu Leu Leu Leu Leu Pro Leu Leu1 5 10 15Trp Leu Leu Val Leu Thr Pro Gly Arg Pro Ala Ala Gly Leu Ser Thr 20 25 30Cys Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg Ile Glu Ala 35 40 45Ile Arg Gly Gln Ile Leu Ser Lys Leu Arg Leu Ala Ser Pro Pro Ser 50 55 60Gln Gly Glu Val Pro Pro Gly Pro Leu Pro Glu Ala Val Leu Ala Leu65 70 75 80Tyr Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser Ala Glu Pro Glu 85 90 95Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys Glu Val Thr Arg Val Leu 100 105 110Met Val Glu Thr His Asn Glu Ile Tyr Asp Lys Phe Lys Gln Ser Thr 115 120 125His Ser Ile Tyr Met Phe Phe Asn Thr Ser Glu Leu Arg Glu Ala Val 130 135 140Pro Glu Pro Val Leu Leu Ser Arg Ala Glu Leu Arg Leu Leu Arg Leu145 150 155 160Lys Leu Lys Val Glu Gln His Val Glu Leu Tyr Gln Lys Tyr Ser Asn 165 170 175Asn Ser Trp Arg Tyr Leu Ser Asn Arg Leu Leu Ala Pro Ser Asp Ser 180 185 190Pro Glu Trp Leu Ser Phe Asp Val Thr Gly Val Val Arg Gln Trp Leu 195 200 205Ser Arg Gly Gly Glu Ile Glu Gly Phe Arg Leu Ser Ala His Cys Ser 210 215 220Cys Asp Ser Arg Asp Asn Thr Leu Gln Val Asp Ile Asn Gly Phe Thr225 230 235 240Thr Gly Arg Arg Gly Asp Leu Ala Thr Ile His Gly Met Asn Arg Pro 245 250 255Phe Leu Leu Leu Met Ala Thr Pro Leu Glu Arg Ala Gln His Leu Gln 260 265 270Ser Ser Arg His Arg Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser 275 280 285Thr Glu Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys 290 295 300Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys Gly Tyr His Ala Asn305 310 315 320Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr 325 330 335Ser Lys Val Leu Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser Ala 340 345 350Ala Pro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr 355 360 365Tyr Val Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn Met Ile Val 370 375 380Arg Ser Cys Lys Cys Ser385 39010499PRTHomo sapiens 104Met Lys Val Ser Ala Ala Leu Leu Cys Leu Leu Leu Ile Ala Ala Thr1 5 10 15Phe Ile Pro Gln Gly Leu Ala Gln Pro Asp Ala Ile Asn Ala Pro Val 20 25 30Thr Cys Cys Tyr Asn Phe Thr Asn Arg Lys Ile Ser Val Gln Arg Leu 35 40 45Ala Ser Tyr Arg Arg Ile Thr Ser Ser Lys Cys Pro Lys Glu Ala Val 50 55 60Ile Phe Lys Thr Ile Val Ala Lys Glu Ile Cys Ala Asp Pro Lys Gln65 70 75 80Lys Trp Val Gln Asp Ser Met Asp His Leu Asp Lys Gln Thr Gln Thr 85 90 95Pro Lys Thr105798PRTHomo sapiens 105Met Asn Leu Gln Pro Ile Phe Trp Ile Gly Leu Ile Ser Ser Val Cys1 5 10 15Cys Val Phe Ala Gln Thr Asp Glu Asn Arg Cys Leu Lys Ala Asn Ala 20 25 30Lys Ser Cys Gly Glu Cys Ile Gln Ala Gly Pro Asn Cys Gly Trp Cys 35 40 45Thr Asn Ser Thr Phe Leu Gln Glu Gly Met Pro Thr Ser Ala Arg Cys 50 55 60Asp Asp Leu Glu Ala Leu Lys Lys Lys Gly Cys Pro Pro Asp Asp Ile65 70 75 80Glu Asn Pro Arg Gly Ser Lys Asp Ile Lys Lys Asn Lys Asn Val Thr 85 90 95Asn Arg Ser Lys Gly Thr Ala Glu Lys Leu Lys Pro Glu Asp Ile Thr 100 105 110Gln Ile Gln Pro Gln Gln Leu Val Leu Arg Leu Arg Ser Gly Glu Pro 115 120 125Gln Thr Phe Thr Leu Lys Phe Lys Arg Ala Glu Asp Tyr Pro Ile Asp 130 135 140Leu Tyr Tyr Leu Met Asp Leu Ser Tyr Ser Met Lys Asp Asp Leu Glu145 150 155 160Asn Val Lys Ser Leu Gly Thr Asp Leu Met Asn Glu Met Arg Arg Ile 165 170 175Thr Ser Asp Phe Arg Ile Gly Phe Gly Ser Phe Val Glu Lys Thr Val 180 185 190Met Pro Tyr Ile Ser Thr Thr Pro Ala Lys Leu Arg Asn Pro Cys Thr 195 200 205Ser Glu Gln Asn Cys Thr Ser Pro Phe Ser Tyr Lys Asn Val Leu Ser 210 215 220Leu Thr Asn Lys Gly Glu Val Phe Asn Glu Leu Val Gly Lys Gln Arg225 230 235 240Ile Ser Gly Asn Leu Asp Ser Pro Glu Gly Gly Phe Asp Ala Ile Met 245 250 255Gln Val Ala Val Cys Gly Ser Leu Ile Gly Trp Arg Asn Val Thr Arg 260 265 270Leu Leu Val Phe Ser Thr Asp Ala Gly Phe His Phe Ala Gly Asp Gly 275 280 285Lys Leu Gly Gly Ile Val Leu Pro Asn Asp Gly Gln Cys His Leu Glu 290 295 300Asn Asn Met Tyr Thr Met Ser His Tyr Tyr Asp Tyr Pro Ser Ile Ala305 310 315 320His Leu Val Gln Lys Leu Ser Glu Asn Asn Ile Gln Thr Ile Phe Ala 325 330 335Val Thr Glu Glu Phe Gln Pro Val Tyr Lys Glu Leu Lys Asn Leu Ile 340 345 350Pro Lys Ser Ala Val Gly Thr Leu Ser Ala Asn Ser Ser Asn Val Ile 355 360 365Gln Leu Ile Ile Asp Ala Tyr Asn Ser Leu Ser Ser Glu Val Ile Leu 370 375 380Glu Asn Gly Lys Leu Ser Glu Gly Val Thr Ile Ser Tyr Lys Ser Tyr385 390 395 400Cys Lys Asn Gly Val Asn Gly Thr Gly Glu Asn Gly Arg Lys Cys Ser 405 410 415Asn Ile Ser Ile Gly Asp Glu Val Gln Phe Glu Ile Ser Ile Thr Ser 420 425 430Asn Lys Cys Pro Lys Lys Asp Ser Asp Ser Phe Lys Ile Arg Pro Leu 435 440 445Gly Phe Thr Glu Glu Val Glu Val Ile Leu Gln Tyr Ile Cys Glu Cys 450 455 460Glu Cys Gln Ser Glu Gly Ile Pro Glu Ser Pro

Lys Cys His Glu Gly465 470 475 480Asn Gly Thr Phe Glu Cys Gly Ala Cys Arg Cys Asn Glu Gly Arg Val 485 490 495Gly Arg His Cys Glu Cys Ser Thr Asp Glu Val Asn Ser Glu Asp Met 500 505 510Asp Ala Tyr Cys Arg Lys Glu Asn Ser Ser Glu Ile Cys Ser Asn Asn 515 520 525Gly Glu Cys Val Cys Gly Gln Cys Val Cys Arg Lys Arg Asp Asn Thr 530 535 540Asn Glu Ile Tyr Ser Gly Lys Phe Cys Glu Cys Asp Asn Phe Asn Cys545 550 555 560Asp Arg Ser Asn Gly Leu Ile Cys Gly Gly Asn Gly Val Cys Lys Cys 565 570 575Arg Val Cys Glu Cys Asn Pro Asn Tyr Thr Gly Ser Ala Cys Asp Cys 580 585 590Ser Leu Asp Thr Ser Thr Cys Glu Ala Ser Asn Gly Gln Ile Cys Asn 595 600 605Gly Arg Gly Ile Cys Glu Cys Gly Val Cys Lys Cys Thr Asp Pro Lys 610 615 620Phe Gln Gly Gln Thr Cys Glu Met Cys Gln Thr Cys Leu Gly Val Cys625 630 635 640Ala Glu His Lys Glu Cys Val Gln Cys Arg Ala Phe Asn Lys Gly Glu 645 650 655Lys Lys Asp Thr Cys Thr Gln Glu Cys Ser Tyr Phe Asn Ile Thr Lys 660 665 670Val Glu Ser Arg Asp Lys Leu Pro Gln Pro Val Gln Pro Asp Pro Val 675 680 685Ser His Cys Lys Glu Lys Asp Val Asp Asp Cys Trp Phe Tyr Phe Thr 690 695 700Tyr Ser Val Asn Gly Asn Asn Glu Val Met Val His Val Val Glu Asn705 710 715 720Pro Glu Cys Pro Thr Gly Pro Asp Ile Ile Pro Ile Val Ala Gly Val 725 730 735Val Ala Gly Ile Val Leu Ile Gly Leu Ala Leu Leu Leu Ile Trp Lys 740 745 750Leu Leu Met Ile Ile His Asp Arg Arg Glu Phe Ala Lys Phe Glu Lys 755 760 765Glu Lys Met Asn Ala Lys Trp Asp Thr Gly Glu Asn Pro Ile Tyr Lys 770 775 780Ser Ala Val Thr Thr Val Val Asn Pro Lys Tyr Glu Gly Lys785 790 795106156PRTHomo sapiens 106Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ile Leu Ala Leu1 5 10 15Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Lys Lys 20 25 30Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu 35 40 45Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr 50 55 60Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys Gln Leu Gln65 70 75 80Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala 85 90 95Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile 100 105 110Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys 115 120 125Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp 130 135 140Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr145 150 155107986PRTHomo sapiens 107Met Pro Gly Val Ala Arg Leu Pro Leu Leu Leu Gly Leu Leu Leu Leu1 5 10 15Pro Arg Pro Gly Arg Pro Leu Asp Leu Ala Asp Tyr Thr Tyr Asp Leu 20 25 30Ala Glu Glu Asp Asp Ser Glu Pro Leu Asn Tyr Lys Asp Pro Cys Lys 35 40 45Ala Ala Ala Phe Leu Gly Asp Ile Ala Leu Asp Glu Glu Asp Leu Arg 50 55 60Ala Phe Gln Val Gln Gln Ala Val Asp Leu Arg Arg His Thr Ala Arg65 70 75 80Lys Ser Ser Ile Lys Ala Ala Val Pro Gly Asn Thr Ser Thr Pro Ser 85 90 95Cys Gln Ser Thr Asn Gly Gln Pro Gln Arg Gly Ala Cys Gly Arg Trp 100 105 110Arg Gly Arg Ser Arg Ser Arg Arg Ala Ala Thr Ser Arg Pro Glu Arg 115 120 125Val Trp Pro Asp Gly Val Ile Pro Phe Val Ile Gly Gly Asn Phe Thr 130 135 140Gly Ser Gln Arg Ala Val Phe Arg Gln Ala Met Arg His Trp Glu Lys145 150 155 160His Thr Cys Val Thr Phe Leu Glu Arg Thr Asp Glu Asp Ser Tyr Ile 165 170 175Val Phe Thr Tyr Arg Pro Cys Gly Cys Cys Ser Tyr Val Gly Arg Arg 180 185 190Gly Gly Gly Pro Gln Ala Ile Ser Ile Gly Lys Asn Cys Asp Lys Phe 195 200 205Gly Ile Val Val His Glu Leu Gly His Val Val Gly Phe Trp His Glu 210 215 220His Thr Arg Pro Asp Arg Asp Arg His Val Ser Ile Val Arg Glu Asn225 230 235 240Ile Gln Pro Gly Gln Glu Tyr Asn Phe Leu Lys Met Glu Pro Gln Glu 245 250 255Val Glu Ser Leu Gly Glu Thr Tyr Asp Phe Asp Ser Ile Met His Tyr 260 265 270Ala Arg Asn Thr Phe Ser Arg Gly Ile Phe Leu Asp Thr Ile Val Pro 275 280 285Lys Tyr Glu Val Asn Gly Val Lys Pro Pro Ile Gly Gln Arg Thr Arg 290 295 300Leu Ser Lys Gly Asp Ile Ala Gln Ala Arg Lys Leu Tyr Lys Cys Pro305 310 315 320Ala Cys Gly Glu Thr Leu Gln Asp Ser Thr Gly Asn Phe Ser Ser Pro 325 330 335Glu Tyr Pro Asn Gly Tyr Ser Ala His Met His Cys Val Trp Arg Ile 340 345 350Ser Val Thr Pro Gly Glu Lys Ile Ile Leu Asn Phe Thr Ser Leu Asp 355 360 365Leu Tyr Arg Ser Arg Leu Cys Trp Tyr Asp Tyr Val Glu Val Arg Asp 370 375 380Gly Phe Trp Arg Lys Ala Pro Leu Arg Gly Arg Phe Cys Gly Ser Lys385 390 395 400Leu Pro Glu Pro Ile Val Ser Thr Asp Ser Arg Leu Trp Val Glu Phe 405 410 415Arg Ser Ser Ser Asn Trp Val Gly Lys Gly Phe Phe Ala Val Tyr Glu 420 425 430Ala Ile Cys Gly Gly Asp Val Lys Lys Asp Tyr Gly His Ile Gln Ser 435 440 445Pro Asn Tyr Pro Asp Asp Tyr Arg Pro Ser Lys Val Cys Ile Trp Arg 450 455 460Ile Gln Val Ser Glu Gly Phe His Val Gly Leu Thr Phe Gln Ser Phe465 470 475 480Glu Ile Glu Arg His Asp Ser Cys Ala Tyr Asp Tyr Leu Glu Val Arg 485 490 495Asp Gly His Ser Glu Ser Ser Thr Leu Ile Gly Arg Tyr Cys Gly Tyr 500 505 510Glu Lys Pro Asp Asp Ile Lys Ser Thr Ser Ser Arg Leu Trp Leu Lys 515 520 525Phe Val Ser Asp Gly Ser Ile Asn Lys Ala Gly Phe Ala Val Asn Phe 530 535 540Phe Lys Glu Val Asp Glu Cys Ser Arg Pro Asn Arg Gly Gly Cys Glu545 550 555 560Gln Arg Cys Leu Asn Thr Leu Gly Ser Tyr Lys Cys Ser Cys Asp Pro 565 570 575Gly Tyr Glu Leu Ala Pro Asp Lys Arg Arg Cys Glu Ala Ala Cys Gly 580 585 590Gly Phe Leu Thr Lys Leu Asn Gly Ser Ile Thr Ser Pro Gly Trp Pro 595 600 605Lys Glu Tyr Pro Pro Asn Lys Asn Cys Ile Trp Gln Leu Val Ala Pro 610 615 620Thr Gln Tyr Arg Ile Ser Leu Gln Phe Asp Phe Phe Glu Thr Glu Gly625 630 635 640Asn Asp Val Cys Lys Tyr Asp Phe Val Glu Val Arg Ser Gly Leu Thr 645 650 655Ala Asp Ser Lys Leu His Gly Lys Phe Cys Gly Ser Glu Lys Pro Glu 660 665 670Val Ile Thr Ser Gln Tyr Asn Asn Met Arg Val Glu Phe Lys Ser Asp 675 680 685Asn Thr Val Ser Lys Lys Gly Phe Lys Ala His Phe Phe Ser Asp Lys 690 695 700Asp Glu Cys Ser Lys Asp Asn Gly Gly Cys Gln Gln Asp Cys Val Asn705 710 715 720Thr Phe Gly Ser Tyr Glu Cys Gln Cys Arg Ser Gly Phe Val Leu His 725 730 735Asp Asn Lys His Asp Cys Lys Glu Ala Gly Cys Asp His Lys Val Thr 740 745 750Ser Thr Ser Gly Thr Ile Thr Ser Pro Asn Trp Pro Asp Lys Tyr Pro 755 760 765Ser Lys Lys Glu Cys Thr Trp Ala Ile Ser Ser Thr Pro Gly His Arg 770 775 780Val Lys Leu Thr Phe Met Glu Met Asp Ile Glu Ser Gln Pro Glu Cys785 790 795 800Ala Tyr Asp His Leu Glu Val Phe Asp Gly Arg Asp Ala Lys Ala Pro 805 810 815Val Leu Gly Arg Phe Cys Gly Ser Lys Lys Pro Glu Pro Val Leu Ala 820 825 830Thr Gly Ser Arg Met Phe Leu Arg Phe Tyr Ser Asp Asn Ser Val Gln 835 840 845Arg Lys Gly Phe Gln Ala Ser His Ala Thr Glu Cys Gly Gly Gln Val 850 855 860Arg Ala Asp Val Lys Thr Lys Asp Leu Tyr Ser His Ala Gln Phe Gly865 870 875 880Asp Asn Asn Tyr Pro Gly Gly Val Asp Cys Glu Trp Val Ile Val Ala 885 890 895Glu Glu Gly Tyr Gly Val Glu Leu Val Phe Gln Thr Phe Glu Val Glu 900 905 910Glu Glu Thr Asp Cys Gly Tyr Asp Tyr Met Glu Leu Phe Asp Gly Tyr 915 920 925Asp Ser Thr Ala Pro Arg Leu Gly Arg Tyr Cys Gly Ser Gly Pro Pro 930 935 940Glu Glu Val Tyr Ser Ala Gly Asp Ser Val Leu Val Lys Phe His Ser945 950 955 960Asp Asp Thr Ile Thr Lys Lys Gly Phe His Leu Arg Tyr Thr Ser Thr 965 970 975Lys Phe Gln Asp Thr Leu His Ser Arg Lys 980 985108148PRTHomo sapiens 108Met Arg Gly Ser Glu Leu Pro Leu Val Leu Leu Ala Leu Val Leu Cys1 5 10 15Leu Ala Pro Arg Gly Arg Ala Val Pro Leu Pro Ala Gly Gly Gly Thr 20 25 30Val Leu Thr Lys Met Tyr Pro Arg Gly Asn His Trp Ala Val Gly His 35 40 45Leu Met Gly Lys Lys Ser Thr Gly Glu Ser Ser Ser Val Ser Glu Arg 50 55 60Gly Ser Leu Lys Gln Gln Leu Arg Glu Tyr Ile Arg Trp Glu Glu Ala65 70 75 80Ala Arg Asn Leu Leu Gly Leu Ile Glu Ala Lys Glu Asn Arg Asn His 85 90 95Gln Pro Pro Gln Pro Lys Ala Leu Gly Asn Gln Gln Pro Ser Trp Asp 100 105 110Ser Glu Asp Ser Ser Asn Phe Lys Asp Val Gly Ser Lys Gly Lys Val 115 120 125Gly Arg Leu Ser Ala Pro Gly Ser Gln Arg Glu Gly Arg Asn Pro Gln 130 135 140Leu Asn Gln Gln145109170PRTHomo sapiens 109Met Asp Thr Arg Asn Lys Ala Gln Leu Leu Val Leu Leu Thr Leu Leu1 5 10 15Ser Val Leu Phe Ser Gln Thr Ser Ala Trp Pro Leu Tyr Arg Ala Pro 20 25 30Ser Ala Leu Arg Leu Gly Asp Arg Ile Pro Phe Glu Gly Ala Asn Glu 35 40 45Pro Asp Gln Val Ser Leu Lys Glu Asp Ile Asp Met Leu Gln Asn Ala 50 55 60Leu Ala Glu Asn Asp Thr Pro Tyr Tyr Asp Val Ser Arg Asn Ala Arg65 70 75 80His Ala Asp Gly Val Phe Thr Ser Asp Phe Ser Lys Leu Leu Gly Gln 85 90 95Leu Ser Ala Lys Lys Tyr Leu Glu Ser Leu Met Gly Lys Arg Val Ser 100 105 110Ser Asn Ile Ser Glu Asp Pro Val Pro Val Lys Arg His Ser Asp Ala 115 120 125Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln Met Ala Val Lys 130 135 140Lys Tyr Leu Asn Ser Ile Leu Asn Gly Lys Arg Ser Ser Glu Gly Glu145 150 155 160Ser Pro Asp Phe Pro Glu Glu Leu Glu Lys 165 170110110PRTHomo sapiens 110Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu1 5 10 15Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn Gln His Leu Cys Gly 20 25 30Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe 35 40 45Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly 50 55 60Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu65 70 75 80Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys 85 90 95Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn 100 105 110111153PRTHomo sapiens 111Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe1 5 10 15Cys Asp Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His Leu 20 25 30Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala 35 40 45Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 50 55 60Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly65 70 75 80Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys 85 90 95Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 100 105 110Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp 115 120 125Met Pro Lys Thr Gln Lys Glu Val His Leu Lys Asn Ala Ser Arg Gly 130 135 140Ser Ala Gly Asn Lys Asn Tyr Arg Met145 150112195PRTHomo sapiens 112Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe1 5 10 15Cys Asp Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His Leu 20 25 30Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala 35 40 45Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe 50 55 60Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly65 70 75 80Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys 85 90 95Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu 100 105 110Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp 115 120 125Met Pro Lys Thr Gln Lys Tyr Gln Pro Pro Ser Thr Asn Lys Asn Thr 130 135 140Lys Ser Gln Arg Arg Lys Gly Trp Pro Lys Thr His Pro Gly Gly Glu145 150 155 160Gln Lys Glu Gly Thr Glu Ala Ser Leu Gln Ile Arg Gly Lys Lys Lys 165 170 175Glu Gln Arg Arg Glu Ile Gly Ser Arg Asn Ala Glu Cys Arg Gly Lys 180 185 190Lys Gly Lys 195113141PRTHomo sapiens 113Met Gly Phe Gln Lys Phe Ser Pro Phe Leu Ala Leu Ser Ile Leu Val1 5 10 15Leu Leu Gln Ala Gly Ser Leu His Ala Ala Pro Phe Arg Ser Ala Leu 20 25 30Glu Ser Ser Pro Ala Asp Pro Ala Thr Leu Ser Glu Asp Glu Ala Arg 35 40 45Leu Leu Leu Ala Ala Leu Val Gln Asp Tyr Val Gln Met Lys Ala Ser 50 55 60Glu Leu Glu Gln Glu Gln Glu Arg Glu Gly Ser Ser Leu Asp Ser Pro65 70 75 80Arg Ser Lys Arg Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr 85 90 95Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro Gln Thr Ala Ile Gly 100 105 110Val Gly Ala Pro Gly Lys Lys Arg Asp Met Ser Ser Asp Leu Glu Arg 115 120 125Asp His Arg Pro His Val Ser Met Pro Gln Asn Ala Asn 130 135 140114554PRTHomo sapiens 114Met Thr Ala Pro Gly Ala Ala Gly Arg Cys Pro Pro Thr Thr Trp Leu1 5 10 15Gly Ser Leu Leu Leu Leu Val Cys Leu Leu Ala Ser Arg Ser Ile Thr 20 25 30Glu Glu Val Ser Glu Tyr Cys Ser His Met Ile Gly Ser Gly His Leu 35 40 45Gln Ser Leu Gln Arg Leu Ile Asp Ser Gln Met Glu Thr Ser Cys Gln 50 55 60Ile Thr Phe Glu Phe Val

Asp Gln Glu Gln Leu Lys Asp Pro Val Cys65 70 75 80Tyr Leu Lys Lys Ala Phe Leu Leu Val Gln Asp Ile Met Glu Asp Thr 85 90 95Met Arg Phe Arg Asp Asn Thr Pro Asn Ala Ile Ala Ile Val Gln Leu 100 105 110Gln Glu Leu Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp Tyr Glu 115 120 125Glu His Asp Lys Ala Cys Val Arg Thr Phe Tyr Glu Thr Pro Leu Gln 130 135 140Leu Leu Glu Lys Val Lys Asn Val Phe Asn Glu Thr Lys Asn Leu Leu145 150 155 160Asp Lys Asp Trp Asn Ile Phe Ser Lys Asn Cys Asn Asn Ser Phe Ala 165 170 175Glu Cys Ser Ser Gln Asp Val Val Thr Lys Pro Asp Cys Asn Cys Leu 180 185 190Tyr Pro Lys Ala Ile Pro Ser Ser Asp Pro Ala Ser Val Ser Pro His 195 200 205Gln Pro Leu Ala Pro Ser Met Ala Pro Val Ala Gly Leu Thr Trp Glu 210 215 220Asp Ser Glu Gly Thr Glu Gly Ser Ser Leu Leu Pro Gly Glu Gln Pro225 230 235 240Leu His Thr Val Asp Pro Gly Ser Ala Lys Gln Arg Pro Pro Arg Ser 245 250 255Thr Cys Gln Ser Phe Glu Pro Pro Glu Thr Pro Val Val Lys Asp Ser 260 265 270Thr Ile Gly Gly Ser Pro Gln Pro Arg Pro Ser Val Gly Ala Phe Asn 275 280 285Pro Gly Met Glu Asp Ile Leu Asp Ser Ala Met Gly Thr Asn Trp Val 290 295 300Pro Glu Glu Ala Ser Gly Glu Ala Ser Glu Ile Pro Val Pro Gln Gly305 310 315 320Thr Glu Leu Ser Pro Ser Arg Pro Gly Gly Gly Ser Met Gln Thr Glu 325 330 335Pro Ala Arg Pro Ser Asn Phe Leu Ser Ala Ser Ser Pro Leu Pro Ala 340 345 350Ser Ala Lys Gly Gln Gln Pro Ala Asp Val Thr Gly Thr Ala Leu Pro 355 360 365Arg Val Gly Pro Val Arg Pro Thr Gly Gln Asp Trp Asn His Thr Pro 370 375 380Gln Lys Thr Asp His Pro Ser Ala Leu Leu Arg Asp Pro Pro Glu Pro385 390 395 400Gly Ser Pro Arg Ile Ser Ser Leu Arg Pro Gln Gly Leu Ser Asn Pro 405 410 415Ser Thr Leu Ser Ala Gln Pro Gln Leu Ser Arg Ser His Ser Ser Gly 420 425 430Ser Val Leu Pro Leu Gly Glu Leu Glu Gly Arg Arg Ser Thr Arg Asp 435 440 445Arg Arg Ser Pro Ala Glu Pro Glu Gly Gly Pro Ala Ser Glu Gly Ala 450 455 460Ala Arg Pro Leu Pro Arg Phe Asn Ser Val Pro Leu Thr Asp Thr Gly465 470 475 480His Glu Arg Gln Ser Glu Gly Ser Ser Ser Pro Gln Leu Gln Glu Ser 485 490 495Val Phe His Leu Leu Val Pro Ser Val Ile Leu Val Leu Leu Ala Val 500 505 510Gly Gly Leu Leu Phe Tyr Arg Trp Arg Arg Arg Ser His Gln Glu Pro 515 520 525Gln Arg Ala Asp Ser Pro Leu Glu Gln Pro Glu Gly Ser Pro Leu Thr 530 535 540Gln Asp Asp Arg Gln Val Glu Leu Pro Val545 55011520PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 115Gly Thr Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys1 5 10 15Ser Ala Glu Asp 2011620PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 116Gly Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp1 5 10 15Thr Glu Glu Tyr 2011720PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 117Gly Thr Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Lys1 5 10 15Thr Glu Glu Tyr 2011820PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 118Gly Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Lys1 5 10 15Thr Glu Glu Tyr 2011920PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 119Gly Arg Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Arg1 5 10 15Thr Glu Glu Tyr 2012020PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 120Gly Thr Phe Phe Tyr Gly Gly Ser Arg Gly Arg Arg Asn Asn Phe Arg1 5 10 15Thr Glu Glu Tyr 2012120PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 121Cys Thr Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys1 5 10 15Ser Ala Glu Asp 2012220PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 122Cys Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp1 5 10 15Thr Glu Glu Tyr 2012320PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 123Cys Thr Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Lys1 5 10 15Thr Glu Glu Tyr 2012420PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 124Cys Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Lys1 5 10 15Thr Glu Glu Tyr 2012520PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 125Cys Arg Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Arg1 5 10 15Thr Glu Glu Tyr 2012620PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 126Cys Thr Phe Phe Tyr Gly Gly Ser Arg Gly Arg Arg Asn Asn Phe Arg1 5 10 15Thr Glu Glu Tyr 2012720PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 127Thr Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser1 5 10 15Ala Glu Asp Gly 2012820PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 128Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr1 5 10 15Glu Glu Tyr Gly 2012920PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 129Thr Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Lys Thr1 5 10 15Glu Glu Tyr Gly 2013020PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 130Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Lys Thr1 5 10 15Glu Glu Tyr Gly 2013120PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 131Arg Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Arg Thr1 5 10 15Glu Glu Tyr Gly 2013220PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 132Thr Phe Phe Tyr Gly Gly Ser Arg Gly Arg Arg Asn Asn Phe Arg Thr1 5 10 15Glu Glu Tyr Gly 2013320PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 133Thr Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser1 5 10 15Ala Glu Asp Cys 2013420PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 134Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr1 5 10 15Glu Glu Tyr Cys 2013520PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 135Thr Phe Phe Tyr Gly Gly Cys Arg Gly Lys Arg Asn Asn Phe Lys Thr1 5 10 15Glu Glu Tyr Cys 2013620PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 136Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Lys Thr1 5 10 15Glu Glu Tyr Cys 2013720PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 137Arg Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Arg Thr1 5 10 15Glu Glu Tyr Cys 2013820PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 138Thr Phe Phe Tyr Gly Gly Ser Arg Gly Arg Arg Asn Asn Phe Arg Thr1 5 10 15Glu Glu Tyr Cys 201391296PRTClostridium botulinum 139Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Val Gly Gln Met Gln Pro 20 25 30Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr65 70 75 80Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155 160Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn225 230 235 240Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305 310 315 320Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys 435 440 445Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu465 470 475 480Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu545 550 555 560His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu 565 570 575Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys 580 585 590Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 610 615 620Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala625 630 635 640Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645 650 655Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala 660 665 670Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys 675 680 685Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys705 710 715 720Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu 725 730 735Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770 775 780Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met785 790 795 800Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys 805 810 815Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly 820 825 830Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp 835 840 845Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser 850 855 860Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn865 870 875 880Leu Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser 885 890 895Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn 900 905 910Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu 915 920 925Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser 930 935 940Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn945 950 955 960Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val 965 970 975Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu 980 985 990Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser 995 1000 1005Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg 1010 1015 1020Leu Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln 1025 1030 1035Lys Pro Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile 1040 1045 1050Met Phe Lys Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp 1055 1060 1065Ile Lys Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu 1070 1075 1080Ile Lys Asp Leu Tyr Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys 1085 1090 1095Asp Phe Trp Gly Asp Tyr Leu Gln Tyr Asp Lys Pro Tyr Tyr Met 1100 1105 1110Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn Asn Val 1115 1120 1125Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg Gly Ser Val 1130 1135 1140Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr Arg Gly Thr 1145 1150 1155Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn Lys Asp Asn Ile 1160 1165 1170Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val Val Val Lys Asn 1175 1180 1185Lys Glu Tyr Arg Leu Ala

Thr Asn Ala Ser Gln Ala Gly Val Glu 1190 1195 1200Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser 1205 1210 1215Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn 1220 1225 1230Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala 1250 1255 1260Ser Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu 1265 1270 1275Gly Cys Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu 1280 1285 1290Arg Pro Leu 12951401291PRTClostridium botulinum 140Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn1 5 10 15Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn65 70 75 80Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe 85 90 95Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn 130 135 140Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile145 150 155 160Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly 165 170 175Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln 180 185 190Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr225 230 235 240Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255Phe Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270Gly Gly Gln Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser Ile 275 280 285Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300Lys Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr305 310 315 320Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340 345 350Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375 380Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile385 390 395 400Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405 410 415Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr 420 425 430Lys Ile Gln Met Cys Lys Ser Val Lys Ala Pro Gly Ile Cys Ile Asp 435 440 445Val Asp Asn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser 450 455 460Asp Asp Leu Ser Lys Asn Glu Arg Ile Glu Tyr Asn Thr Gln Ser Asn465 470 475 480Tyr Ile Glu Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp 485 490 495Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys 515 520 525Lys Ile Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln 530 535 540Thr Phe Pro Leu Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp545 550 555 560Asp Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590Trp Val Lys Gln Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605Asn Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615 620Gly Leu Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu625 630 635 640Asn Ala Phe Glu Ile Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro 645 650 655Glu Leu Leu Ile Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile 660 665 670Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys 675 680 685Arg Asn Glu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val Ala Gln Trp 690 695 700Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr705 710 715 720Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr 725 730 735Arg Tyr Asn Ile Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile Asp 740 745 750Phe Asn Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln Ala Ile 755 760 765Asp Asn Ile Asn Asn Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met 770 775 780Lys Lys Met Ile Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn785 790 795 800Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805 810 815Leu Ile Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu 820 825 830Lys Thr Ile Met Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile 835 840 845Leu Ile Glu Met Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile 850 855 860Ile Leu Asn Leu Arg Tyr Lys Asp Asn Asn Leu Ile Asp Leu Ser Gly865 870 875 880Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys 885 890 895Asn Gln Phe Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr 900 905 910Gln Asn Gln Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser Val 915 920 925Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn 930 935 940Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser945 950 955 960Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile 965 970 975Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg 980 985 990Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr 995 1000 1005Asn Asn Leu Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu 1010 1015 1020Ser Asn Thr Asp Ile Lys Asp Ile Arg Glu Val Ile Ala Asn Gly 1025 1030 1035Glu Ile Ile Phe Lys Leu Asp Gly Asp Ile Asp Arg Thr Gln Phe 1040 1045 1050Ile Trp Met Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu Ser Gln 1055 1060 1065Ser Asn Ile Glu Glu Arg Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr 1070 1075 1080Leu Lys Asp Phe Trp Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr 1085 1090 1095Tyr Met Phe Asn Ala Gly Asn Lys Asn Ser Tyr Ile Lys Leu Lys 1100 1105 1110Lys Asp Ser Pro Val Gly Glu Ile Leu Thr Arg Ser Lys Tyr Asn 1115 1120 1125Gln Asn Ser Lys Tyr Ile Asn Tyr Arg Asp Leu Tyr Ile Gly Glu 1130 1135 1140Lys Phe Ile Ile Arg Arg Lys Ser Asn Ser Gln Ser Ile Asn Asp 1145 1150 1155Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr Leu Asp Phe Phe Asn 1160 1165 1170Leu Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys Tyr Phe Lys Lys 1175 1180 1185Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp Ser Asp Glu 1190 1195 1200Phe Tyr Asn Thr Ile Gln Ile Lys Glu Tyr Asp Glu Gln Pro Thr 1205 1210 1215Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp 1220 1225 1230Glu Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Ile 1235 1240 1245Val Phe Glu Glu Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr 1250 1255 1260Leu Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys 1265 1270 1275Asn Trp Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1280 1285 12901411291PRTClostridium botulinum 141Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn1 5 10 15Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp65 70 75 80Ser Asp Lys Asp Pro Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145 150 155 160Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly225 230 235 240Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly305 310 315 320Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385 390 395 400Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430Phe Thr Lys Phe Cys His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn 435 440 445Lys Thr Leu Asp Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450 455 460Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys465 470 475 480Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser 485 490 495Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu 500 505 510Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515 520 525Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu 530 535 540Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu545 550 555 560Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala 565 570 575Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly 580 585 590Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp 595 600 605Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp 610 615 620Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn625 630 635 640Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys 675 680 685Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser 690 695 700Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe705 710 715 720Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly 725 730 735Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775 780Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile785 790 795 800Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn 805 810 815Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu 820 825 830Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile 835 840 845Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860Phe Asn Asn Ile Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Arg Lys865 870 875 880Asn Thr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 885 890 895Gly Asp Val Gln Leu Asn Pro Ile Phe Pro Phe Asp Phe Lys Leu Gly 900 905 910Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr Gln Asn Glu Asn 915 920 925Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser Phe Trp Ile 930 935 940Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr Ile Ile Asp945 950 955 960Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly Ile Ile Ser Asn Phe 965 970 975Leu Val Phe Thr Leu Lys Gln Asn Glu Asp Ser Glu Gln Ser Ile Asn 980 985 990Phe Ser Tyr Asp Ile Ser Asn Asn Ala Pro Gly Tyr Asn Lys Trp Phe 995 1000 1005Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys Ile Tyr 1010 1015 1020Ile Asn Gly Lys Leu Ile Asp Thr Ile Lys Val Lys Glu Leu Thr 1025 1030 1035Gly Ile Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile Asn Lys Ile 1040 1045 1050Pro Asp Thr Gly Leu Ile Thr Ser Asp Ser

Asp Asn Ile Asn Met 1055 1060 1065Trp Ile Arg Asp Phe Tyr Ile Phe Ala Lys Glu Leu Asp Gly Lys 1070 1075 1080Asp Ile Asn Ile Leu Phe Asn Ser Leu Gln Tyr Thr Asn Val Val 1085 1090 1095Lys Asp Tyr Trp Gly Asn Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr 1100 1105 1110Met Val Asn Ile Asp Tyr Leu Asn Arg Tyr Met Tyr Ala Asn Ser 1115 1120 1125Arg Gln Ile Val Phe Asn Thr Arg Arg Asn Asn Asn Asp Phe Asn 1130 1135 1140Glu Gly Tyr Lys Ile Ile Ile Lys Arg Ile Arg Gly Asn Thr Asn 1145 1150 1155Asp Thr Arg Val Arg Gly Gly Asp Ile Leu Tyr Phe Asp Met Thr 1160 1165 1170Ile Asn Asn Lys Ala Tyr Asn Leu Phe Met Lys Asn Glu Thr Met 1175 1180 1185Tyr Ala Asp Asn His Ser Thr Glu Asp Ile Tyr Ala Ile Gly Leu 1190 1195 1200Arg Glu Gln Thr Lys Asp Ile Asn Asp Asn Ile Ile Phe Gln Ile 1205 1210 1215Gln Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Gln Ile Phe Lys 1220 1225 1230Ser Asn Phe Asn Gly Glu Asn Ile Ser Gly Ile Cys Ser Ile Gly 1235 1240 1245Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His Asn Tyr 1250 1255 1260Leu Val Pro Thr Val Lys Gln Gly Asn Tyr Ala Ser Leu Leu Glu 1265 1270 1275Ser Thr Ser Thr His Trp Gly Phe Val Pro Val Ser Glu 1280 1285 12901421276PRTClostridium botulinum 142Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp1 5 10 15Asn Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu Ile Thr Thr 20 25 30Pro Val Lys Ala Phe Met Ile Thr Gln Asn Ile Trp Val Ile Pro Glu 35 40 45Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro 50 55 60Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr Asp65 70 75 80Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys Leu Phe Lys Arg 85 90 95Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn Tyr Leu Val Val 100 105 110Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp 115 120 125Phe Thr Arg His Thr Thr Asn Ile Ala Val Glu Lys Phe Glu Asn Gly 130 135 140Ser Trp Lys Val Thr Asn Ile Ile Thr Pro Ser Val Leu Ile Phe Gly145 150 155 160Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln Gly 165 170 175Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu 180 185 190Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser Asn 195 200 205Gln Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met Asp Pro Val 210 215 220Ile Ala Leu Met His Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly225 230 235 240Ile Asn Ile Pro Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly 245 250 255Phe Phe Ser Gln Asp Gly Pro Asn Val Gln Phe Glu Glu Leu Tyr Thr 260 265 270Phe Gly Gly Leu Asp Val Glu Ile Ile Pro Gln Ile Glu Arg Ser Gln 275 280 285Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu 290 295 300Asn Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp305 310 315 320Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys Asp Asn 325 330 335Thr Gly Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser Leu Tyr Ser 340 345 350Asp Leu Thr Asn Val Met Ser Glu Val Val Tyr Ser Ser Gln Tyr Asn 355 360 365Val Lys Asn Arg Thr His Tyr Phe Ser Arg His Tyr Leu Pro Val Phe 370 375 380Ala Asn Ile Leu Asp Asp Asn Ile Tyr Thr Ile Arg Asp Gly Phe Asn385 390 395 400Leu Thr Asn Lys Gly Phe Asn Ile Glu Asn Ser Gly Gln Asn Ile Glu 405 410 415Arg Asn Pro Ala Leu Gln Lys Leu Ser Ser Glu Ser Val Val Asp Leu 420 425 430Phe Thr Lys Val Cys Leu Arg Leu Thr Lys Asn Ser Arg Asp Asp Ser 435 440 445Thr Cys Ile Lys Val Lys Asn Asn Arg Leu Pro Tyr Val Ala Asp Lys 450 455 460Asp Ser Ile Ser Gln Glu Ile Phe Glu Asn Lys Ile Ile Thr Asp Glu465 470 475 480Thr Asn Val Gln Asn Tyr Ser Asp Lys Phe Ser Leu Asp Glu Ser Ile 485 490 495Leu Asp Gly Gln Val Pro Ile Asn Pro Glu Ile Val Asp Pro Leu Leu 500 505 510Pro Asn Val Asn Met Glu Pro Leu Asn Leu Pro Gly Glu Glu Ile Val 515 520 525Phe Tyr Asp Asp Ile Thr Lys Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr 530 535 540Tyr Leu Glu Ser Gln Lys Leu Ser Asn Asn Val Glu Asn Ile Thr Leu545 550 555 560Thr Thr Ser Val Glu Glu Ala Leu Gly Tyr Ser Asn Lys Ile Tyr Thr 565 570 575Phe Leu Pro Ser Leu Ala Glu Lys Val Asn Lys Gly Val Gln Ala Gly 580 585 590Leu Phe Leu Asn Trp Ala Asn Glu Val Val Glu Asp Phe Thr Thr Asn 595 600 605Ile Met Lys Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Val Ile 610 615 620Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Ser Ala Leu Arg625 630 635 640Gly Asn Phe Asn Gln Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu 645 650 655Glu Gly Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly Val Phe Thr Phe 660 665 670Tyr Ser Ser Ile Gln Glu Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn 675 680 685Cys Leu Glu Gln Arg Val Lys Arg Trp Lys Asp Ser Tyr Gln Trp Met 690 695 700Val Ser Asn Trp Leu Ser Arg Ile Thr Thr Gln Phe Asn His Ile Asn705 710 715 720Tyr Gln Met Tyr Asp Ser Leu Ser Tyr Gln Ala Asp Ala Ile Lys Ala 725 730 735Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn 740 745 750Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys Ile 755 760 765Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val 770 775 780Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn785 790 795 800Lys Phe Asp Leu Arg Thr Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser 805 810 815His Asn Ile Ile Leu Val Gly Glu Val Asp Arg Leu Lys Ala Lys Val 820 825 830Asn Glu Ser Phe Glu Asn Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr 835 840 845Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr Phe Asn Ser Ile 850 855 860Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Lys Lys Asn Ala Leu Val865 870 875 880Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Gly Asp Asn Val Gln 885 890 895Leu Asn Thr Ile Tyr Thr Asn Asp Phe Lys Leu Ser Ser Ser Gly Asp 900 905 910Lys Ile Ile Val Asn Leu Asn Asn Asn Ile Leu Tyr Ser Ala Ile Tyr 915 920 925Glu Asn Ser Ser Val Ser Phe Trp Ile Lys Ile Ser Lys Asp Leu Thr 930 935 940Asn Ser His Asn Glu Tyr Thr Ile Ile Asn Ser Ile Glu Gln Asn Ser945 950 955 960Gly Trp Lys Leu Cys Ile Arg Asn Gly Asn Ile Glu Trp Ile Leu Gln 965 970 975Asp Val Asn Arg Lys Tyr Lys Ser Leu Ile Phe Asp Tyr Ser Glu Ser 980 985 990Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe Val Thr Ile Thr 995 1000 1005Asn Asn Ile Met Gly Tyr Met Lys Leu Tyr Ile Asn Gly Glu Leu 1010 1015 1020Lys Gln Ser Gln Lys Ile Glu Asp Leu Asp Glu Val Lys Leu Asp 1025 1030 1035Lys Thr Ile Val Phe Gly Ile Asp Glu Asn Ile Asp Glu Asn Gln 1040 1045 1050Met Leu Trp Ile Arg Asp Phe Asn Ile Phe Ser Lys Glu Leu Ser 1055 1060 1065Asn Glu Asp Ile Asn Ile Val Tyr Glu Gly Gln Ile Leu Arg Asn 1070 1075 1080Val Ile Lys Asp Tyr Trp Gly Asn Pro Leu Lys Phe Asp Thr Glu 1085 1090 1095Tyr Tyr Ile Ile Asn Asp Asn Tyr Ile Asp Arg Tyr Ile Ala Pro 1100 1105 1110Glu Ser Asn Val Leu Val Leu Val Gln Tyr Pro Asp Arg Ser Lys 1115 1120 1125Leu Tyr Thr Gly Asn Pro Ile Thr Ile Lys Ser Val Ser Asp Lys 1130 1135 1140Asn Pro Tyr Ser Arg Ile Leu Asn Gly Asp Asn Ile Ile Leu His 1145 1150 1155Met Leu Tyr Asn Ser Arg Lys Tyr Met Ile Ile Arg Asp Thr Asp 1160 1165 1170Thr Ile Tyr Ala Thr Gln Gly Gly Glu Cys Ser Gln Asn Cys Val 1175 1180 1185Tyr Ala Leu Lys Leu Gln Ser Asn Leu Gly Asn Tyr Gly Ile Gly 1190 1195 1200Ile Phe Ser Ile Lys Asn Ile Val Ser Lys Asn Lys Tyr Cys Ser 1205 1210 1215Gln Ile Phe Ser Ser Phe Arg Glu Asn Thr Met Leu Leu Ala Asp 1220 1225 1230Ile Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn Ala Tyr Thr Pro 1235 1240 1245Val Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser Thr Ser Ser 1250 1255 1260Phe Trp Lys Phe Ile Ser Arg Asp Pro Gly Trp Val Glu 1265 1270 12751431251PRTClostridium botulinum 143Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg1 5 10 15Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln Glu Phe Tyr Lys Ser 20 25 30Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile 35 40 45Gly Thr Thr Pro Gln Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Glu Glu Lys65 70 75 80Asp Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn 85 90 95Asn Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp 115 120 125Ala Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile Leu 130 135 140Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr145 150 155 160Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175Arg Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190Arg Phe Asn Asp Asn Cys Met Asn Glu Phe Ile Gln Asp Pro Ala Leu 195 200 205Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu225 230 235 240Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly 245 250 255Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr 260 265 270Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys 275 280 285Val Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290 295 300Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn305 310 315 320Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335Phe Asp Leu Arg Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile 340 345 350Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355 360 365Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe 370 375 380Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr385 390 395 400Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410 415Ser Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435 440 445Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala465 470 475 480Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala 485 490 495Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His 500 505 510Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys Val 515 520 525Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala 530 535 540Leu Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile545 550 555 560Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile 565 570 575Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr 580 585 590Val Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu 595 600 605Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala 610 615 620Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu625 630 635 640Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655Ser Asp Asn Lys Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660 665 670Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680 685Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met 690 695 700Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Ile Lys Thr Ile Ile Glu705 710 715 720Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725 730 735Lys Tyr Asp Ile Lys Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser 740 745 750Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser 755 760 765Tyr Leu Met Lys Ile Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770 775 780Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr Ile Ile Gln His785 790 795 800Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Thr 805 810 815Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 835 840 845Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850 855 860Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys865 870 875 880Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895Glu Val Asn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940Asp Asn Asn Ser Gly Trp Lys Val

Ser Leu Asn His Asn Glu Ile Ile945 950 955 960Trp Thr Phe Glu Asp Asn Arg Gly Ile Asn Gln Lys Leu Ala Phe Asn 965 970 975Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe 980 985 990Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn 995 1000 1005Gly Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile 1010 1015 1020His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr 1025 1030 1035Thr Arg Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu 1040 1045 1050Leu Asp Glu Thr Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro Asn 1055 1060 1065Thr Asn Ile Leu Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp 1070 1075 1080Lys Glu Tyr Tyr Leu Leu Asn Val Leu Lys Pro Asn Asn Phe Ile 1085 1090 1095Asp Arg Arg Lys Asp Ser Thr Leu Ser Ile Asn Asn Ile Arg Ser 1100 1105 1110Thr Ile Leu Leu Ala Asn Arg Leu Tyr Ser Gly Ile Lys Val Lys 1115 1120 1125Ile Gln Arg Val Asn Asn Ser Ser Thr Asn Asp Asn Leu Val Arg 1130 1135 1140Lys Asn Asp Gln Val Tyr Ile Asn Phe Val Ala Ser Lys Thr His 1145 1150 1155Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr Thr Asn Lys Glu Lys 1160 1165 1170Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe Asn Gln Val Val 1175 1180 1185Val Met Asn Ser Val Gly Asn Cys Thr Met Asn Phe Lys Asn Asn 1190 1195 1200Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr Val 1205 1210 1215Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His Thr Asn 1220 1225 1230Ser Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly Trp 1235 1240 1245Gln Glu Lys 12501441274PRTClostridium botulinum 144Met Pro Val Ala Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp1 5 10 15Asp Thr Ile Leu Tyr Met Gln Ile Pro Tyr Glu Glu Lys Ser Lys Lys 20 25 30Tyr Tyr Lys Ala Phe Glu Ile Met Arg Asn Val Trp Ile Ile Pro Glu 35 40 45Arg Asn Thr Ile Gly Thr Asn Pro Ser Asp Phe Asp Pro Pro Ala Ser 50 55 60Leu Lys Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr65 70 75 80Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr Ile Lys Leu Phe Lys 85 90 95Arg Ile Asn Ser Asn Pro Ala Gly Lys Val Leu Leu Gln Glu Ile Ser 100 105 110Tyr Ala Lys Pro Tyr Leu Gly Asn Asp His Thr Pro Ile Asp Glu Phe 115 120 125Ser Pro Val Thr Arg Thr Thr Ser Val Asn Ile Lys Leu Ser Thr Asn 130 135 140Val Glu Ser Ser Met Leu Leu Asn Leu Leu Val Leu Gly Ala Gly Pro145 150 155 160Asp Ile Phe Glu Ser Cys Cys Tyr Pro Val Arg Lys Leu Ile Asp Pro 165 170 175Asp Val Val Tyr Asp Pro Ser Asn Tyr Gly Phe Gly Ser Ile Asn Ile 180 185 190Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile Ser Gly 195 200 205Gly His Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala Ile Ser 210 215 220Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu Tyr Gly Ala Arg225 230 235 240Gly Val Thr Tyr Glu Glu Thr Ile Glu Val Lys Gln Ala Pro Leu Met 245 250 255Ile Ala Glu Lys Pro Ile Arg Leu Glu Glu Phe Leu Thr Phe Gly Gly 260 265 270Gln Asp Leu Asn Ile Ile Thr Ser Ala Met Lys Glu Lys Ile Tyr Asn 275 280 285Asn Leu Leu Ala Asn Tyr Glu Lys Ile Ala Thr Arg Leu Ser Glu Val 290 295 300Asn Ser Ala Pro Pro Glu Tyr Asp Ile Asn Glu Tyr Lys Asp Tyr Phe305 310 315 320Gln Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly Ser Tyr Thr Val 325 330 335Asn Glu Asn Lys Phe Asn Glu Ile Tyr Lys Lys Leu Tyr Ser Phe Thr 340 345 350Glu Ser Asp Leu Ala Asn Lys Phe Lys Val Lys Cys Arg Asn Thr Tyr 355 360 365Phe Ile Lys Tyr Glu Phe Leu Lys Val Pro Asn Leu Leu Asp Asp Asp 370 375 380Ile Tyr Thr Val Ser Glu Gly Phe Asn Ile Gly Asn Leu Ala Val Asn385 390 395 400Asn Arg Gly Gln Ser Ile Lys Leu Asn Pro Lys Ile Ile Asp Ser Ile 405 410 415Pro Asp Lys Gly Leu Val Glu Lys Ile Val Lys Phe Cys Lys Ser Val 420 425 430Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu Cys Ile Arg Val 435 440 445Asn Asn Ser Glu Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn Glu 450 455 460Asn Asp Ile Asn Thr Pro Lys Glu Ile Asp Asp Thr Thr Asn Leu Asn465 470 475 480Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val Ile Leu Asp Tyr Asn Ser 485 490 495Gln Thr Ile Pro Gln Ile Ser Asn Arg Thr Leu Asn Thr Leu Val Gln 500 505 510Asp Asn Ser Tyr Val Pro Arg Tyr Asp Ser Asn Gly Thr Ser Glu Ile 515 520 525Glu Glu Tyr Asp Val Val Asp Phe Asn Val Phe Phe Tyr Leu His Ala 530 535 540Gln Lys Val Pro Glu Gly Glu Thr Asn Ile Ser Leu Thr Ser Ser Ile545 550 555 560Asp Thr Ala Leu Leu Glu Glu Ser Lys Asp Ile Phe Phe Ser Ser Glu 565 570 575Phe Ile Asp Thr Ile Asn Lys Pro Val Asn Ala Ala Leu Phe Ile Asp 580 585 590Trp Ile Ser Lys Val Ile Arg Asp Phe Thr Thr Glu Ala Thr Gln Lys 595 600 605Ser Thr Val Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Val 610 615 620Gly Leu Ala Leu Asn Ile Ile Ile Glu Ala Glu Lys Gly Asn Phe Glu625 630 635 640Glu Ala Phe Glu Leu Leu Gly Val Gly Ile Leu Leu Glu Phe Val Pro 645 650 655Glu Leu Thr Ile Pro Val Ile Leu Val Phe Thr Ile Lys Ser Tyr Ile 660 665 670Asp Ser Tyr Glu Asn Lys Asn Lys Ala Ile Lys Ala Ile Asn Asn Ser 675 680 685Leu Ile Glu Arg Glu Ala Lys Trp Lys Glu Ile Tyr Ser Trp Ile Val 690 695 700Ser Asn Trp Leu Thr Arg Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu705 710 715 720Gln Met Tyr Gln Ala Leu Gln Asn Gln Val Asp Ala Ile Lys Thr Ala 725 730 735Ile Glu Tyr Lys Tyr Asn Asn Tyr Thr Ser Asp Glu Lys Asn Arg Leu 740 745 750Glu Ser Glu Tyr Asn Ile Asn Asn Ile Glu Glu Glu Leu Asn Lys Lys 755 760 765Val Ser Leu Ala Met Lys Asn Ile Glu Arg Phe Met Thr Glu Ser Ser 770 775 780Ile Ser Tyr Leu Met Lys Leu Ile Asn Glu Ala Lys Val Gly Lys Leu785 790 795 800Lys Lys Tyr Asp Asn His Val Lys Ser Asp Leu Leu Asn Tyr Ile Leu 805 810 815Asp His Arg Ser Ile Leu Gly Glu Gln Thr Asn Glu Leu Ser Asp Leu 820 825 830Val Thr Ser Thr Leu Asn Ser Ser Ile Pro Phe Glu Leu Ser Ser Tyr 835 840 845Thr Asn Asp Lys Ile Leu Ile Ile Tyr Phe Asn Arg Leu Tyr Lys Lys 850 855 860Ile Lys Asp Ser Ser Ile Leu Asp Met Arg Tyr Glu Asn Asn Lys Phe865 870 875 880Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn Gly Asn Val 885 890 895Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe Gly Ile Tyr Asn Ser Arg 900 905 910Leu Ser Glu Val Asn Ile Ala Gln Asn Asn Asp Ile Ile Tyr Asn Ser 915 920 925Arg Tyr Gln Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Lys His 930 935 940Tyr Lys Pro Met Asn His Asn Arg Glu Tyr Thr Ile Ile Asn Cys Met945 950 955 960Gly Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Arg Thr Val Arg Asp 965 970 975Cys Glu Ile Ile Trp Thr Leu Gln Asp Thr Ser Gly Asn Lys Glu Asn 980 985 990Leu Ile Phe Arg Tyr Glu Glu Leu Asn Arg Ile Ser Asn Tyr Ile Asn 995 1000 1005Lys Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser 1010 1015 1020Arg Ile Tyr Ile Asn Gly Asn Leu Ile Val Glu Lys Ser Ile Ser 1025 1030 1035Asn Leu Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile 1040 1045 1050Val Gly Cys Asp Asp Glu Thr Tyr Val Gly Ile Arg Tyr Phe Lys 1055 1060 1065Val Phe Asn Thr Glu Leu Asp Lys Thr Glu Ile Glu Thr Leu Tyr 1070 1075 1080Ser Asn Glu Pro Asp Pro Ser Ile Leu Lys Asn Tyr Trp Gly Asn 1085 1090 1095Tyr Leu Leu Tyr Asn Lys Lys Tyr Tyr Leu Phe Asn Leu Leu Arg 1100 1105 1110Lys Asp Lys Tyr Ile Thr Leu Asn Ser Gly Ile Leu Asn Ile Asn 1115 1120 1125Gln Gln Arg Gly Val Thr Glu Gly Ser Val Phe Leu Asn Tyr Lys 1130 1135 1140Leu Tyr Glu Gly Val Glu Val Ile Ile Arg Lys Asn Gly Pro Ile 1145 1150 1155Asp Ile Ser Asn Thr Asp Asn Phe Val Arg Lys Asn Asp Leu Ala 1160 1165 1170Tyr Ile Asn Val Val Asp Arg Gly Val Glu Tyr Arg Leu Tyr Ala 1175 1180 1185Asp Thr Lys Ser Glu Lys Glu Lys Ile Ile Arg Thr Ser Asn Leu 1190 1195 1200Asn Asp Ser Leu Gly Gln Ile Ile Val Met Asp Ser Ile Gly Asn 1205 1210 1215Asn Cys Thr Met Asn Phe Gln Asn Asn Asn Gly Ser Asn Ile Gly 1220 1225 1230Leu Leu Gly Phe His Ser Asn Asn Leu Val Ala Ser Ser Trp Tyr 1235 1240 1245Tyr Asn Asn Ile Arg Arg Asn Thr Ser Ser Asn Gly Cys Phe Trp 1250 1255 1260Ser Ser Ile Ser Lys Glu Asn Gly Trp Lys Glu 1265 12701451297PRTClostridium botulinumMOD_RES(7)..(7)Any amino acid 145Met Pro Val Asn Ile Lys Xaa Phe Asn Tyr Asn Asp Pro Ile Asn Asn1 5 10 15Asp Asp Ile Ile Met Met Glu Pro Phe Asn Asp Pro Gly Pro Gly Thr 20 25 30Tyr Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile Val Pro Glu 35 40 45Arg Phe Thr Tyr Gly Phe Gln Pro Asp Gln Phe Asn Ala Ser Thr Gly 50 55 60Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr Leu Lys65 70 75 80Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met Ile Lys Leu Phe 85 90 95Asn Arg Ile Asn Ser Lys Pro Ser Gly Gln Arg Leu Leu Asp Met Ile 100 105 110Val Asp Ala Ile Pro Tyr Leu Gly Asn Ala Ser Thr Pro Pro Asp Lys 115 120 125Phe Ala Ala Asn Val Ala Asn Val Ser Ile Asn Lys Lys Ile Ile Gln 130 135 140Pro Gly Ala Glu Asp Gln Ile Lys Gly Leu Met Thr Asn Leu Ile Ile145 150 155 160Phe Gly Pro Gly Pro Val Leu Ser Asp Asn Phe Thr Asp Ser Met Ile 165 170 175Met Asn Gly His Ser Pro Ile Ser Glu Gly Phe Gly Ala Arg Met Met 180 185 190Ile Arg Phe Cys Pro Ser Cys Leu Asn Val Phe Asn Asn Val Gln Glu 195 200 205Asn Lys Asp Thr Ser Ile Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro 210 215 220Ala Leu Thr Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr225 230 235 240Gly Ile Lys Ile Ser Asn Leu Pro Ile Thr Pro Asn Thr Lys Glu Phe 245 250 255Phe Met Gln His Ser Asp Pro Val Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270Gly Gly His Asp Pro Ser Val Ile Ser Pro Ser Thr Asp Met Asn Ile 275 280 285Tyr Asn Lys Ala Leu Gln Asn Phe Gln Asp Ile Ala Asn Arg Leu Asn 290 295 300Ile Val Ser Ser Ala Gln Gly Ser Gly Ile Asp Ile Ser Leu Tyr Lys305 310 315 320Gln Ile Tyr Lys Asn Lys Tyr Asp Phe Val Glu Asp Pro Asn Gly Lys 325 330 335Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys Ala Leu Met 340 345 350Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu Tyr Gly Ile Lys Thr 355 360 365Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro Ile Lys Thr Glu Lys 370 375 380Leu Leu Asp Asn Thr Ile Tyr Thr Gln Asn Glu Gly Phe Asn Ile Ala385 390 395 400Ser Lys Asn Leu Lys Thr Glu Phe Asn Gly Gln Asn Lys Ala Val Asn 405 410 415Lys Glu Ala Tyr Glu Glu Ile Ser Leu Glu His Leu Val Ile Tyr Arg 420 425 430Ile Ala Met Cys Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu 435 440 445Gln Cys Ile Ile Val Asn Asn Glu Asp Leu Phe Phe Ile Ala Asn Lys 450 455 460Asp Ser Phe Ser Lys Asp Leu Ala Lys Ala Glu Thr Ile Ala Tyr Asn465 470 475 480Thr Gln Asn Asn Thr Ile Glu Asn Asn Phe Ser Ile Asp Gln Leu Ile 485 490 495Leu Asp Asn Asp Leu Ser Ser Gly Ile Asp Leu Pro Asn Glu Asn Thr 500 505 510Glu Pro Phe Thr Asn Phe Asp Asp Ile Asp Ile Pro Val Tyr Ile Lys 515 520 525Gln Ser Ala Leu Lys Lys Ile Phe Val Asp Gly Asp Ser Leu Phe Glu 530 535 540Tyr Leu His Ala Gln Thr Phe Pro Ser Asn Ile Glu Asn Leu Gln Leu545 550 555 560Thr Asn Ser Leu Asn Asp Ala Leu Arg Asn Asn Asn Lys Val Tyr Thr 565 570 575Phe Phe Ser Thr Asn Leu Val Glu Lys Ala Asn Thr Val Val Gly Ala 580 585 590Ser Leu Phe Val Asn Trp Val Lys Gly Val Ile Asp Asp Phe Thr Ser 595 600 605Glu Ser Thr Gln Lys Ser Thr Ile Asp Lys Val Ser Asp Val Ser Ile 610 615 620Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Val Gly Asn Glu Thr Ala625 630 635 640Lys Glu Asn Phe Lys Asn Ala Phe Glu Ile Gly Gly Ala Ala Ile Leu 645 650 655Met Glu Phe Ile Pro Glu Leu Ile Val Pro Ile Val Gly Phe Phe Thr 660 665 670Leu Glu Ser Tyr Val Gly Asn Lys Gly His Ile Ile Met Thr Ile Ser 675 680 685Asn Ala Leu Lys Lys Arg Asp Gln Lys Trp Thr Asp Met Tyr Gly Leu 690 695 700Ile Val Ser Gln Trp Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile705 710 715 720Lys Glu Arg Met Tyr Asn Ala Leu Asn Asn Gln Ser Gln Ala Ile Glu 725 730 735Lys Ile Ile Glu Asp Gln Tyr Asn Arg Tyr Ser Glu Glu Asp Lys Met 740 745 750Asn Ile Asn Ile Asp Phe Asn Asp Ile Asp Phe Lys Leu Asn Gln Ser 755 760 765Ile Asn Leu Ala Ile Asn Asn Ile Asp Asp Phe Ile Asn Gln Cys Ser 770 775 780Ile Ser Tyr Leu Met Asn Arg Met Ile Pro Leu Ala Val Lys Lys Leu785 790 795 800Lys Asp Phe Asp Asp Asn Leu Lys Arg Asp Leu Leu Glu Tyr Ile Asp 805 810 815Thr Asn Glu Leu Tyr Leu Leu Asp Glu Val Asn Ile Leu Lys Ser Lys 820 825 830Val Asn Arg His Leu Lys Asp Ser Ile Pro Phe Asp Leu Ser Leu Tyr 835 840 845Thr Lys Asp Thr Ile Leu Ile Gln Val Phe Asn Asn Tyr Ile Ser Asn 850 855 860Ile Ser Ser Asn Ala Ile Leu Ser Leu Ser Tyr Arg Gly Gly Arg Leu865

870 875 880Ile Asp Ser Ser Gly Tyr Gly Ala Thr Met Asn Val Gly Ser Asp Val 885 890 895Ile Phe Asn Asp Ile Gly Asn Gly Gln Phe Lys Leu Asn Asn Ser Glu 900 905 910Asn Ser Asn Ile Thr Ala His Gln Ser Lys Phe Val Val Tyr Asp Ser 915 920 925Met Phe Asp Asn Phe Ser Ile Asn Phe Trp Val Arg Thr Pro Lys Tyr 930 935 940Asn Asn Asn Asp Ile Gln Thr Tyr Leu Gln Asn Glu Tyr Thr Ile Ile945 950 955 960Ser Cys Ile Lys Asn Asp Ser Gly Trp Lys Val Ser Ile Lys Gly Asn 965 970 975Arg Ile Ile Trp Thr Leu Ile Asp Val Asn Ala Lys Ser Lys Ser Ile 980 985 990Phe Phe Glu Tyr Ser Ile Lys Asp Asn Ile Ser Asp Tyr Ile Asn Lys 995 1000 1005Trp Phe Ser Ile Thr Ile Thr Asn Asp Arg Leu Gly Asn Ala Asn 1010 1015 1020Ile Tyr Ile Asn Gly Ser Leu Lys Lys Ser Glu Lys Ile Leu Asn 1025 1030 1035Leu Asp Arg Ile Asn Ser Ser Asn Asp Ile Asp Phe Lys Leu Ile 1040 1045 1050Asn Cys Thr Asp Thr Thr Lys Phe Val Trp Ile Lys Asp Phe Asn 1055 1060 1065Ile Phe Gly Arg Glu Leu Asn Ala Thr Glu Val Ser Ser Leu Tyr 1070 1075 1080Trp Ile Gln Ser Ser Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn 1085 1090 1095Pro Leu Arg Tyr Asp Thr Gln Tyr Tyr Leu Phe Asn Gln Gly Met 1100 1105 1110Gln Asn Ile Tyr Ile Lys Tyr Phe Ser Lys Ala Ser Met Gly Glu 1115 1120 1125Thr Ala Pro Arg Thr Asn Phe Asn Asn Ala Ala Ile Asn Tyr Gln 1130 1135 1140Asn Leu Tyr Leu Gly Leu Arg Phe Ile Ile Lys Lys Ala Ser Asn 1145 1150 1155Ser Arg Asn Ile Asn Asn Asp Asn Ile Val Arg Glu Gly Asp Tyr 1160 1165 1170Ile Tyr Leu Asn Ile Asp Asn Ile Ser Asp Glu Ser Tyr Arg Val 1175 1180 1185Tyr Val Leu Val Asn Ser Lys Glu Ile Gln Thr Gln Leu Phe Leu 1190 1195 1200Ala Pro Ile Asn Asp Asp Pro Thr Phe Tyr Asp Val Leu Gln Ile 1205 1210 1215Lys Lys Tyr Tyr Glu Lys Thr Thr Tyr Asn Cys Gln Ile Leu Cys 1220 1225 1230Glu Lys Asp Thr Lys Thr Phe Gly Leu Phe Gly Ile Gly Lys Phe 1235 1240 1245Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr Asp Asn Tyr Phe 1250 1255 1260Cys Ile Ser Gln Trp Tyr Leu Arg Arg Ile Ser Glu Asn Ile Asn 1265 1270 1275Lys Leu Arg Leu Gly Cys Asn Trp Gln Phe Ile Pro Val Asp Glu 1280 1285 1290Gly Trp Thr Glu 12951461315PRTClostridium tetani 146Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn1 5 10 15Asp Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile 20 25 30Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu 35 40 45Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser 50 55 60Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr65 70 75 80Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn 85 90 95Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile 100 105 110Asn Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe 115 120 125Asp Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro 130 135 140Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe145 150 155 160Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg Gly Ile Val Leu 165 170 175Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser 180 185 190Ile Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn 195 200 205Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe 210 215 220Gln Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His225 230 235 240Gly Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys 245 250 255Gln Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu 260 265 270Phe Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys 275 280 285Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn 290 295 300Lys Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp305 310 315 320Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser 325 330 335Asn Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn 340 345 350Ser Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn 355 360 365Ile Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys 370 375 380Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe385 390 395 400Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met 405 410 415Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val 420 425 430Ser Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile 435 440 445Arg Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly 450 455 460Glu Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu465 470 475 480Lys Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr 485 490 495Asn Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile 500 505 510Ile Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg 515 520 525Thr Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser 530 535 540Asn Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile545 550 555 560Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile 565 570 575Thr Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile 580 585 590Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln 595 600 605Gly Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr 610 615 620Asn Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser625 630 635 640Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly 645 650 655Tyr Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu 660 665 670Leu Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu 675 680 685Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile 690 695 700Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys705 710 715 720Leu Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys 725 730 735Arg Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile 740 745 750Lys Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys 755 760 765Glu Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu 770 775 780Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser785 790 795 800Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln 805 810 815Leu Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile 820 825 830Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu 835 840 845Ser Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser 850 855 860Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile865 870 875 880Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile 885 890 895Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala 900 905 910Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn 915 920 925Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn 930 935 940Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys945 950 955 960Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile 965 970 975Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser 980 985 990Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala 995 1000 1005Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe 1010 1015 1020Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn 1025 1030 1035Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met 1040 1045 1050Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn 1055 1060 1065Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr 1070 1075 1080Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro 1085 1090 1095Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe 1100 1105 1110Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr 1115 1120 1125Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys 1130 1135 1140Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr 1145 1150 1155Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu 1160 1165 1170Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser 1175 1180 1185Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn 1190 1195 1200Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe 1205 1210 1215Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly 1220 1225 1230Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu 1235 1240 1245Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala 1250 1255 1260Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp 1265 1270 1275Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His 1280 1285 1290Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr 1295 1300 1305Asp Glu Gly Trp Thr Asn Asp 1310 1315147567PRTCorynephage beta 147Met Leu Val Arg Gly Tyr Val Val Ser Arg Lys Leu Phe Ala Ser Ile1 5 10 15Leu Ile Gly Ala Leu Leu Gly Ile Gly Ala Pro Pro Ser Ala His Ala 20 25 30Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn 35 40 45Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln 50 55 60Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp65 70 75 80Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly 85 90 95Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly Val 100 105 110Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val 115 120 125Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu 130 135 140Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly145 150 155 160Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser 165 170 175Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser 180 185 190Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp 195 200 205Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg 210 215 220Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val225 230 235 240Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly 245 250 255Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu 260 265 270Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu 275 280 285His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val 290 295 300Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val305 310 315 320Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu 325 330 335Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala 340 345 350Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser 355 360 365Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp 370 375 380Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe385 390 395 400Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His 405 410 415Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn Thr 420 425 430Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His 435 440 445Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val 450 455 460Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys Thr465 470 475 480His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala Ile 485 490 495Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val Gly 500 505 510Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser Ser 515 520 525Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val Leu 530 535 540Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn Ser Lys Leu Ser545 550 555 560Leu Phe Phe Glu Ile Lys Ser 565148638PRTPseudomonas aeruginosa 148Met His Leu Thr Pro His Trp Ile Pro Leu Val Ala Ser Leu Gly Leu1 5 10 15Leu Ala Gly Gly Ser Phe Ala Ser Ala Ala Glu Glu Ala Phe Asp Leu 20 25 30Trp Asn Glu Cys Ala Lys Ala Cys Val Leu Asp Leu Lys Asp Gly Val 35 40 45Arg Ser Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp Thr Asn Gly 50 55 60Gln Gly Val Leu His Tyr Ser Met Val Leu Glu Gly Gly Asn Asp Ala65 70 75 80Leu Lys Leu Ala Ile Asp Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu 85 90 95Thr Ile Arg Leu Glu Gly Gly Val Glu Pro Asn Lys Pro Val Arg Tyr 100 105 110Ser Tyr Thr Arg Gln Ala Arg Gly Ser Trp Ser Leu Asn Trp Leu Val 115 120 125Pro Ile Gly His Glu Lys Pro Ser Asn Ile Lys Val Phe Ile His Glu 130 135 140Leu Asn

Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr Ile145 150 155 160Glu Met Gly Asp Glu Leu Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe 165 170 175Phe Val Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr Leu Ala Ile 180 185 190Ser His Ala Gly Val Ser Val Val Met Ala Gln Ala Gln Pro Arg Arg 195 200 205Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly Lys Val Leu Cys Leu Leu 210 215 220Asp Pro Leu Asp Gly Val Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn225 230 235 240Leu Asp Asp Thr Trp Glu Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn 245 250 255Pro Ala Lys His Asp Leu Asp Ile Lys Pro Thr Val Ile Ser His Arg 260 265 270Leu His Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln 275 280 285Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg 290 295 300Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val305 310 315 320Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val 325 330 335Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu 340 345 350Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala 355 360 365Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu 370 375 380Ala Gly Ala Ala Ser Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala385 390 395 400Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu 405 410 415Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Ile 420 425 430Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu 435 440 445Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr 450 455 460His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val465 470 475 480Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile 485 490 495Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro 500 505 510Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val 515 520 525Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Gly Leu Thr Leu Ala 530 535 540Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu545 550 555 560Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg 565 570 575Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile 580 585 590Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp 595 600 605Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp 610 615 620Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys625 630 63514919PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 149Gly Glu Phe Val Met Asn Ala Ala Asn Ala Gln Gly His Thr Ala Gly1 5 10 15Thr Arg Leu15010PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 150Thr Gln Ile Glu Asn Leu Lys Glu Lys Gly1 5 101515PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 151Cys Ser Lys Cys Gly1 51528PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 152Cys Phe Thr Lys Trp Phe Phe Cys1 51538PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 153Thr Phe Thr Lys Trp Phe Phe Phe1 515425DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 154gccagacuuu guuggauuug aaatt 2515527RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 155aauuucaaau ccaacaaagu cuggcuu 2715625DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 156ggagggcuuu cuuuguguau uugcc 2515727RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 157ggcaaauaca caaagaaagc ccucccc 271588PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 158Arg Arg Arg Arg Arg Arg Arg Arg1 51599PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 159Arg Arg Arg Arg Arg Arg Arg Arg Arg1 516085PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 160Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Tyr Gly Gly Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Xaa Xaa 8516119PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 161Gly Lys Ser Val Lys Ala Pro Gly Ile Gly Gly Lys Ser Val Lys Ala1 5 10 15Pro Gly Ile

Claims

1. An isolated double stranded ribonucleic acid (dsRNA) composition comprising a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus wherein said first strand and said second strand have a length that is at least 16 and at most 50 nucleotides in length, and a peptide, wherein said peptide has a net charge of about +5 or less and wherein the peptide is conjugated to said dsRNA.

2. An isolated double stranded ribonucleic acid (dsRNA) composition comprising a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus wherein said first strand and said second strand have a length that is at least 16 and at most 50 nucleotides in length, and a peptide, wherein the peptide has no net charge and wherein said peptide is conjugated to said dsRNA.

3. An isolated double stranded ribonucleic acid (dsRNA) composition comprising a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus wherein said first strand and said second strand have a length that is at least 16 and at most 50 nucleotides in length, and a peptide, wherein said peptide has a net charge of about +5 or less and wherein said peptide has at least one anionic amino acid residue. and wherein said peptide is conjugated to said dsRNA.

4. The isolated composition of claim 3 wherein said anionic amino acid is glutamic acid or aspartic acid.

5. An isolated double stranded ribonucleic acid (dsRNA) composition comprising a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus wherein said first strand and said second strand have a length that is at least 6 and at most 19 nucleotides in length, and a peptide, wherein the peptide has a net charge of about +4 or less and wherein said peptide is conjugated to said dsRNA.

6. The isolated composition of claim 1, 2, 3 or 5 wherein said first strand and said second strand have a length that is at least 25 and at most 35 nucleotides, at least 19 and at most 35 nucleotides, at least 19 and at most 24 nucleotides, at least 25 and at most 30 nucleotides, at least 26 and at most 30 nucleotides or at least 21 and a most 23 nucleotides.

7. The isolated composition of claim 1, 2, 3 or 5 wherein said second strand comprises an overhang at the 3' terminus.

8. The isolated composition of claim 1, 2, 3 or 5 wherein said first strand comprises an overhang at the 3' terminus.

9. The isolated composition of claim 1, 2, 3 or 5 wherein at least one of said second strand and said first strand comprises an overhang at the 3' terminus.

10. The isolated composition of claims 9, wherein said nucleotides of said 3' overhang of said first and/or second strand comprise a modified nucleotide.

11. The isolated composition of claims 9, wherein said 3' overhang(s) is/are 1-5 nucleotides in length.

12. The isolated composition of claim 1, 2, 3 or 5 wherein each of said first and second strands consists of the same number of nucleotide residues.

13. The isolated composition of claim 12, wherein the ultimate residue of said 5' terminus of said first strand and the ultimate residue of said 3' terminus of said second strand form a mismatched base pair.

14. The isolated composition of claim 12, wherein the ultimate residue of said 3' terminus of said first strand and the ultimate residue of said 5' terminus of said second strand form a mismatched base pair.

15. The isolated composition of claim 12 wherein the ultimate and penultimate residues of said 5' terminus of said first strand and the ultimate and penultimate residues of said 3' terminus of said second strand form two mismatched base pairs.

16. The isolated composition of claim 12 wherein the ultimate and penultimate residues of said 3' terminus of said first strand and the ultimate and penultimate residues of said 5' terminus of said second strand form two mismatched base pairs.

17. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide comprises 6-50 amino acids.

18. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide comprises 10-50 amino acids.

19. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide comprises 15-30 amino acids.

20. The isolated composition of claim 1, 2, 3 or 5, wherein said peptide comprises up to 10 amino acids.

21. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide has a net charge of about +2 or less.

22. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide has a net charge of about +1 or less.

23. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide comprises one or more proline residues.

24. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide comprises one or more hydrophobic amino acid residues.

25. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide comprises five or more cationic amino acid residues.

26. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide comprises four cationic amino acid residues.

27. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide comprises three cationic amino acid residues.

28. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide comprises two cationic amino acid residues.

29. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide comprises one cationic amino acid residue.

30. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide has no cationic amino acid residues.

31. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide is conjugated to said dsRNA with a stable linker.

32. The isolated composition of claim 31, wherein said stable linker comprises a homobifunctional crosslinker.

33. The isolated composition of claim 31, wherein said stable linker comprises a hetero-bifunctional crosslinker.

34. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide is conjugated to said dsRNA with a cleavable linker.

35. The isolated composition of claim 34, wherein said cleavable linker comprises a disulfide linker.

36. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide is conjugated to said dsRNA with a carbon linker.

37. The isolated composition of claim 36, where said carbon linker comprises no more than eighteen carbons

38. The isolated composition of claim 36, wherein said carbon linker comprises 6 carbons.

39. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide and said dsRNA are conjugated without a linker.

40. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide is conjugated to the 3' end of the first strand of said dsRNA.

41. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide is conjugated to the 3' end of said second strand of said dsRNA.

42. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide is conjugated to the 5' end of the first strand of said dsRNA.

43. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide is conjugated to the 5' end of said second strand of said dsRNA.

44. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide is conjugated to the 5' end of the first strand and the 5' end of said second strand of said dsRNA.

45. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide is conjugated to the 5' end of said first strand and said 3' end of said second strand of said dsRNA.

46. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide is conjugated to the 3' end of the first strand and the 3' end of said second strand of said dsRNA.

47. The isolated composition of claim 1, 2, 3 or 5 wherein said peptide is conjugated to the 3' end of said first strand and said 5' end of said second strand of said dsRNA.

48. The isolated composition of claim 1, 2, 3 or 5 wherein at least one peptide is conjugated internally to said first strand of said dsRNA.

49. The isolated composition of claim 1, 2, 3 or 5 wherein at least one peptide is conjugated internally to said second strand of said dsRNA.

50. The isolated composition of claim 1, 2, 3 or 5 wherein at least one peptide is conjugated internally to said first strand and wherein at least one peptide is conjugated internally to said second strand of said dsRNA.

51. The isolated composition of claim 1, 2, 3 or 5 wherein at least two peptides are conjugated to said dsRNA.

52. The isolated composition of claim 51, wherein said at least two peptides are identical.

53. The isolated composition of claim 51, wherein said at least two peptides are not identical.

54. The isolated composition of claim 1, 2, 3 or 5 further comprising at least one dye molecule, and wherein said dye molecule is conjugated to at least one of said dsRNA and said peptide.

55. The isolated composition of claim 54, wherein said dye molecule is polyaromatic.

56. The isolated composition of claim 54, wherein said dye is a fluorescent dye.

57. The isolated composition of claim 1, 2, 3 or 5 further comprising a therapeutic agent.

58. The isolated composition of claim 57, wherein said therapeutic agent is an anticancer drug.

59. The isolated composition of claim 58, wherein said anticancer drug is selected from the group consisting of paclitaxel, tamoxifen, cisplatin, doxorubicin and vinblastine.

60. The composition of claim 58, wherein said therapeutic agent is a drug to treat a metabolic disease or disorder.

61. The composition of claim 1, 2, 3 or 5, further comprising at least one targeting peptide.

62. The isolated composition of claim 1, 2, 3 or 5, wherein said peptide comprises a portion of a translocation domain of a toxin.

63. The isolated composition of claim 62, wherein said neurotoxin is a clostridial neurotoxin.

64. The isolated composition of claim 1, 2, 3 or 5, wherein starting from the first nucleotide (position 1) at the 3' terminus of the first oligonucleotide strand of said dsRNA, position 1, 2 and/or 3 is/are substituted with a modified nucleotide.

65. The isolated dsRNA of claim 64 wherein said modified nucleotide is a deoxyribonucleotide.

66. The isolated dsRNA of claim 1, 2, 3 or 5, wherein one or both of the first and second oligonucleotide strands comprises a 5' phosphate.

67. The isolated composition of claim 1, 2, 3 or 5, wherein at least one nucleotide of said first or second strand is modified.

68. The isolated composition of claim 67 wherein said modified nucleotide residues are selected from the group consisting of 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O--[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, 2'-amino and 2'-O--(N-methylcarbamate).

69. The isolated composition of claim 1, 2, 3 or 5, wherein said dsRNA is cleaved endogenously in said cell by Dicer.

70. The isolated composition of claim 1, 2, 3 or 5, wherein the amount of said isolated double stranded nucleic acid sufficient to reduce expression of the target gene is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less, 100 picomolar or less, 50 picomolar or less, 20 picomolar or less and 10 picomolar or less in the environment of said cell.

71. The isolated composition of claim 1, 2, 3 or 5, wherein the first and second strands are joined by a chemical linker.

72. The isolated composition of claim 1, 2, 3 or 5, wherein said 3' terminus of said first strand and said 5' terminus of said second strand are joined by a chemical linker.

73. The isolated composition of claim 1, 2, 3 or 5, wherein a nucleotide of said second or first strand is substituted with a modified nucleotide that directs the orientation of Dicer cleavage.

74. The isolated composition of claim 1, 2, 3 or 5, comprising a modified nucleotide selected from the group consisting of a deoxyribonucleotide, a dideoxyribonucleotide, an acyclonucleotide, a 3'-deoxyadenosine (cordycepin), a 3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxyinosine (ddI), a 2',3'-dideoxy-3'-thiacytidine (3TC), a 2',3'-didehydro-2',3'-dideoxythymidine (d4T), a monophosphate nucleotide of 3'-azido-3'-deoxythymidine (AZT), a 2',3'-dideoxy-3'-thiacytidine (3TC) and a monophosphate nucleotide of 2',3'-didehydro-2',3'-dideoxythymidine (d4T), a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2'-O-alkyl ribonucleotide, a 2'-O-methyl ribonucleotide, a 2'-amino ribonucleotide, a 2'-fluoro ribonucleotide, and a locked nucleic acid.

75. The isolated composition of claim 1, 2, 3 or 5 comprising a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.

76. The isolated composition of claim 64, wherein said modified nucleotide residue of said 3' terminus of said first strand is selected from the group consisting of a deoxyribonucleotide, an acyclonucleotide and a fluorescent molecule.

77. The isolated composition of claim 1, 2, 3 or 5, wherein at least one of said nucleotides of said first strand and at least one of said nucleotides of said second strand form a mismatched base pair.

78. The isolated composition of claim 1, 2, 3 or 5, wherein said peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-89.

79. A method for reducing expression of a target gene in a cell, comprising: contacting a cell with said isolated composition as claimed in claim 1, 2, 3 or 5, in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA.

80. A method for selectively inhibiting the growth of a cell comprising contacting a cell with an amount of said isolated composition of claim 1, 2, 3 or 5 sufficient to inhibit the growth of the cell.

81. A method for reducing expression of a target gene in an animal, comprising: treating an animal with said isolated composition as claimed in claim 1, 2, 3 or 5, in an amount effective to reduce expression of a target gene in a cell of the animal in comparison to a reference dsRNA.

82. The method of claim 81, wherein said isolated composition possesses enhanced pharmacokinetics when compared to an appropriate control dsRNA.

83. The method of claim 81, wherein said dsRNA possesses enhanced pharmacodynamics when compared to an appropriate control dsRNA.

84. The method of claim 81, wherein said dsRNA possesses reduced toxicity when compared to an appropriate control dsRNA.

85. The method of claim 81, wherein said dsRNA possesses enhanced intracellular uptake when compared to an appropriate control dsRNA.

86. A pharmaceutical composition for reducing expression of a target gene in a cell of a subject comprising said isolated composition of claim 1, 2, 3 or 5 in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA and a pharmaceutically acceptable carrier.

87. A method of synthesizing a dsRNA-peptide conjugate as claimed in any one of claims 1-78, comprising chemically or enzymatically synthesizing said dsRNA.

88. A kit comprising the dsRNA-peptide conjugate of any one of claims 1-74 and instructions for its use.